Aptamers against Clostridium difficile

ABSTRACT

Compositions comprising aptamers capable of specifically binding to a surface protein of  Clostridium difficile  spore are provided. A method for detecting, enriching, separating, and/or isolating  Clostridium difficile  spores is provided.

RELATED APPLICATIONS

This application claims the benefit of and the priority to U.S.Provisional application Ser. No. 62/857,639, filed Jun. 5, 2019 andProvisional application Ser. No. 62/983,095, filed Feb. 28, 2020, theentire disclosure of each of which is herein incorporated by referencein their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing, submitted herewithwhich includes the file 193519-010105_ST25.txt having the following size25,602 bytes, which was created on Jun. 5, 2020, the contents of whichare hereby incorporated by reference herein.

FIELD OF THE INVENTION

Embodiments of the present invention relate to aptamers thatspecifically bind to a Clostridium difficile spore and methods of usingthe same. For example, embodiments of the invention relate to methods ofdetecting the presence, absence or amount of C. difficile bacteria e.g.spores in a sample using the aptamers described herein.

BACKGROUND TO THE INVENTION

Clostridium difficile (also referred to as C. difficile) is aGram-positive, anaerobic spore former and is an important nosocomial andcommunity-acquired pathogenic bacterium. C. difficile infections (CDI)are a leading cause of infections worldwide with elevated rates ofmorbidity and mortality. Given the rise in antibiotic resistance and thepotential mortality associated with C. difficile infection, controlmeasures are of the highest importance.

SUMMARY

Some aspects of the disclosure relate to an aptamer having a specificbinding affinity for a surface protein of Clostridium difficile spore,wherein the surface protein is a spore coat surface protein or anexosporium layer protein.

In some embodiments, the surface protein is CdeC, CdeM, CotA, CotE orCotE Chitinase. In some embodiments, the surface protein is CdeC havingan amino acid sequence as set forth in SEQ ID NO 18. In someembodiments, the surface protein is CdeM having an amino acid sequenceas set forth in SEQ ID NO: 19. In some embodiments, the surface proteinis CotA having an amino acid sequence as set forth in SEQ ID NO: 15. Insome embodiments, the surface protein is CotE having an amino acidsequence as set forth in SEQ ID NO: 16. In some embodiments, the surfaceprotein is CotE Chitinase protein having an amino acid sequence as setforth in SEQ ID NO: 17.

In some embodiments, the aptamer comprises a nucleic acid sequencehaving at least 90% identity with any one of SEQ ID NOs: 1 to 14 or SEQID NOs: 23-26. In some embodiments, the aptamer comprises a nucleic acidsequence having at least about 30 consecutive nucleotides of a sequencehaving at least 90% identity with any one of SEQ ID NOs: 1 to 14 or SEQID NOs: 23-26.

In some embodiments, the aptamer is a single stranded DNA aptamer.

In some embodiments, the aptamer comprises a detectable label. In someembodiments, the detectable label comprises a fluorophore, ananoparticle, a quantum dot, an enzyme, a radioactive isotope, apre-defined sequence portion, a biotin, a desthiobiotin, a thiol group,an amine group, an azide, an aminoallyl group, a digoxigenin, anantibody, a catalyst, a colloidal metallic particle, a colloidalnon-metallic particle, an organic polymer, a latex particle, ananofiber, a nanotube, a dendrimer, a protein, a liposome, orcombination thereof.

In some embodiments, a composition comprising at least one aptamer isprovided. In some embodiments, the composition comprises at least one ofwater, salt, buffer, detergent, and bovine serum albumin (BSA).

Some aspects of the disclosure relate to a complex comprising (a) anaptamer having a specific binding affinity for a surface protein of aClostridium difficile spore, wherein the surface protein is a spore coatsurface protein or an exosporium layer protein, and (b) a detectablemolecule. In some embodiments, the surface protein is CdeC, CdeM, CotA,CotE or CotE Chitinase

Some aspects of the disclosure relate to a biosensor or test stripcomprising an aptamer having a specific binding affinity for a surfaceprotein of Clostridium difficile spore, wherein the surface protein is aspore coat surface protein or an exosporium layer protein. In someembodiments, the surface protein is CdeC, CdeM, CotA, CotE or CotEChitinase. In some embodiments, the aptamer comprises a nucleic acidsequence having at least 90% identity with any one of the nucleic acidsequences as set forth in any of SEQ ID NOs: 1 to 14 or SEQ ID NOs:23-26. In some embodiments, the aptamer comprises a nucleic acidsequence having at least about 30 consecutive nucleotides of a sequencehaving at least 90% identity with any one of SEQ ID NOs: 1 to 14 or SEQID NOs: 23-26.

Other aspects of the disclosure relate to an apparatus for detecting thepresence, absence or level of Clostridium difficile spores in a sample,the apparatus comprising a support, and an aptamer having a specificbinding affinity for a surface protein of Clostridium difficile spore,wherein the surface protein is a spore coat surface protein or anexosporium layer protein. In some embodiments, the surface protein isCdeC, CdeM, CotA, CotE or CotE Chitinase. In some embodiments, theaptamer comprises a nucleic acid sequence having at least 90% identitywith any one of the nucleic acid sequences as set forth in any of SEQ IDNOs: 1 to 14 or SEQ ID NOs: 23-26. In some embodiments, the aptamercomprises a nucleic acid sequence having at least about 30 consecutivenucleotides of a sequence having at least 90% identity with any one ofSEQ ID NOs: 1 to 14 or SEQ ID NOs: 23-26. In some embodiments, thesample is a sample obtained from a subject suspected of having ordiagnosed with a Clostridium difficile infection or an object located ina hospital environment. In some embodiments, the apparatus is suitablefor surface plasmon resonance (SPR), biolayer interferometry (BLI),lateral flow assay and/or enzyme-linked oligonucleotide assay (ELONA).

In some aspects of the disclosure, there is provided a use of anaptamer, a complex, a composition, a biosensor or test strip, or anapparatus as described herein for detecting, enriching, separatingand/or isolating Clostridium difficile spores.

In some aspects of the disclosure, there is provided a method ofdetecting the presence, absence or amount of Clostridium difficile in asample, the method comprising interacting the sample with an aptamer, acomplex, a composition as described herein, and detecting the presence,absence or amount of Clostridium difficile.

Some aspects of the disclosure relate to a method of visualizingClostridium difficile spores on a surface, comprising contacting asurface with an aptamer having a specific binding affinity for a surfaceprotein of Clostridium difficile spore, wherein the surface protein is aspore coat surface protein or an exosporium layer protein, andvisualizing the presence or absence of C. difficile spores on thesurface. In some embodiments, the surface protein is CdeC, CdeM, CotA,CotE or CotE Chitinase. In some embodiments, the contacting comprisescontacting the surface for a predetermined period of time sufficient toenable the aptamer to bind to a Clostridium difficile spore.

In some embodiments, the method further comprises washing of the surfaceafter the contacting to remove unbound aptamer.

In some embodiments, the method further comprises visualizing theaptamer bound to a Clostridium difficile spore, thereby detecting theClostridium difficile spore.

In some embodiments, the aptamer comprises a nucleic acid sequencehaving at least 90% identity with any one of the nucleic acid sequencesas set forth in any of SEQ ID NOs: 1 to 14 or SEQ ID NOs: 23-26. In someembodiments, the aptamer comprises a nucleic acid sequence having atleast about 30 consecutive nucleotides of a sequence having at least 90%identity with any one of SEQ ID NOs: 1 to 14 or SEQ ID NOs: 23-26.

In some embodiments, the aptamer is conjugated to a detectable moietythereby forming an aptamer conjugate. In some embodiments, thedetectable moiety is a fluorophore. In some embodiments, the fluorophoreemits at a wavelength of between about 500 nm and 510 nm.

In some embodiments, the method further comprises illuminating thesurface with a light source. In some embodiments, light from the lightsource has a predetermined wavelength, and the predetermined wavelengthcorresponds to a wavelength of light emitted by the detectable moiety ofthe aptamer conjugate. In some embodiments, the light source isconfigured to produce light at a wavelength of between about 485 nm and515 nm. In some embodiments, the method further comprises filtering thelight produced by the light source. In some embodiments, the methodcomprises passing the light produced from the light source through abandpass filter.

In some embodiments, the method further comprises photographing alocation on the surface and detecting the presence or absence of theconjugated aptamer bound to Clostridium difficile spores.

Other aspects of the disclosure relate to a kit for visualizingClostridium difficile spores, the kit comprising (a) an aptamercomprising a detectable moiety, the aptamer having a specific bindingaffinity for a surface protein of Clostridium difficile spore, whereinthe surface protein is a spore coat surface protein or an exosporiumlayer protein, (b) a light source, and (c) viewing goggles.

In some embodiments, the surface protein is CdeC, CdeM, CotA, CotE orCotE Chitinase.

In some embodiments, the kit further comprises a bandpass filter.

In some embodiments, the detectable moiety is a fluorophore that emits awavelength of between about 485 nm to 515 nm, the light source isconfigured to produce a light having a wavelength of between about 485nm to 515 nm, and the viewing goggles are orange viewing goggles.

In some embodiments, the light source produces light having a wavelengthof about 505 nm.

In some embodiments, the bandpass filter is a 590 nm bandpass filter.

In some embodiments, the kit further comprises a wash solution to removeunbound aptamers.

In some embodiments, the aptamer is comprised in a buffer solution.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Embodiments of the present invention will be better understood when readin conjunction with the appended drawings. For the purposes ofillustrating the invention, there are shown in the drawings embodimentswhich may be preferred. It is understood, however, that the invention isnot limited to the precise arrangements and instrumentalities shown.

FIG. 1 shows the amino acid sequence (SEQ ID NO: 18) of Clostridiumdifficile CdeC protein.

FIG. 2 shows the amino acid sequence (SEQ ID NO: 19) of Clostridiumdifficile CdeM protein.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 15) of Clostridiumdifficile CotA protein.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 16) of Clostridiumdifficile CotE protein.

FIG. 5A shows the amino acid sequence (SEQ ID NO: 20) of Clostridiumdifficile rCotE protein (N281-F712) C-terminal His-tagged (MW 48,722Da).

FIG. 5B shows the amino acid sequence (SEQ ID NO: 17) of Clostridiumdifficile rCotEC chitinase protein (P381-F712) C-terminal His-tagged (MW36,875 Da).

FIG. 6 shows aptamer recovery following sequential selection rounds inan assay comparing aptamer recovery from CotA loaded beads (left side ofeach data set) with aptamer recovery from blank beads (right side ofeach data set) according to some embodiments of the present disclosure.

FIG. 7 shows aptamer recovery following sequential selection rounds inan assay comparing aptamer recovery from CdeC loaded beads (left side ofeach data set) with aptamer recovery from blank beads (right side ofeach data set) according to some embodiments of the present disclosure.

FIG. 8 shows aptamer recovery following sequential selection rounds inan assay comparing aptamer recovery from CdeM loaded beads (left side ofeach data set) with aptamer recovery from blank beads (right side ofeach data set) according to some embodiments of the present disclosure.

FIG. 9 shows aptamer recovery following sequential selection rounds inan assay comparing aptamer recovery from CotE loaded beads (left side ofeach data set) with aptamer recovery from blank beads (right side ofeach data set according to some embodiments of the present disclosure.

FIG. 10 shows aptamer recovery following sequential selection rounds inan assay comparing aptamer recovery from CotEC Chitinase loaded beads(left side of each data set) with aptamer recovery from blank beads(right side of each data set) according to some embodiments of thepresent disclosure.

FIG. 11 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CotA and the naïve library (Naive Aptamer Pool) orimmobilized CotA and the refined aptamer population (CotA Aptamer Pool)according to some embodiments of the present disclosure.

FIG. 12 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CdeC and the naïve library (Naive Aptamer Pool) orimmobilized CdeC and the refined aptamer population (CdeC Aptamer Pool)according to some embodiments of the present disclosure.

FIG. 13 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CdeM and the naïve library (Naive Aptamer Pool) orimmobilized CdeM and the refined aptamer population (CdeM Aptamer Pool)according to some embodiments of the present disclosure.

FIG. 14 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CotE and the naïve library (Naive Aptamer Pool) orimmobilized CotE and the refined aptamer population (rCotEC AptamerPool) according to some embodiments of the present disclosure.

FIG. 15 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CotEC Chitinase and the naïve library (Naive AptamerPool) or immobilized CotEC Chitinase and the refined aptamer population(CotEC Aptamer Pool) according to some embodiments of the presentdisclosure.

FIG. 16 shows the % aptamer recovery from sequential rounds ofspore-based selection; comparing aptamer recovery from Clostridiumdifficile spores (CD) with recovery from Bacillus subtilis spores (BS)for each of the aptamer populations (named for their proteintarget—CotA) according to some embodiments of the present disclosure.

FIG. 17 shows the % aptamer recovery from sequential rounds ofspore-based selection; comparing aptamer recovery from Clostridiumdifficile spores (CD) with recovery from Bacillus subtilis spores (BS)for each of the aptamer populations (named for their proteintarget—CdeC) according to some embodiments of the present disclosure.

FIG. 18 shows the % aptamer recovery from sequential rounds ofspore-based selection; comparing aptamer recovery from Clostridiumdifficile spores (CD) with recovery from Bacillus subtilis spores (BS)for each of the aptamer populations (named for their proteintarget—CdeM) according to some embodiments of the present disclosure.

FIG. 19 shows the % aptamer recovery from sequential rounds ofspore-based selection; comparing aptamer recovery from Clostridiumdifficile spores (CD) with recovery from Bacillus subtilis spores (BS)for each of the aptamer populations (named for their proteintarget—CotE) according to some embodiments of the present disclosure.

FIG. 20 shows the % aptamer recovery from sequential rounds ofspore-based selection; comparing aptamer recovery from Clostridiumdifficile spores (CD) with recovery from Bacillus subtilis spores (BS)for each of the aptamer populations (named for their proteintarget—CotEC chitinase) according to some embodiments of the presentdisclosure.

FIG. 21 shows brightfield images (left) and epifluoresence images(right) according to some embodiments of the present disclosure. C.difficile spores are localised to the upper images and B. subtilisspores are localised to the lower images. Epifluorescence images oflocalised C. difficile spores (upper right) show spots of fluorescencewhich colocalise with C. difficile spores, which is indicative ofbinding of the fluorescently labeled aptamer population selected againstCotA to the C. difficile spores. Spores showing fluorescent signals arehighlighted with circles for clarity. For comparison, epifluorescenceimages of localised Bacillus subtilis spores (lower right) are shown.The black box signifies dust on the lens.

FIG. 22 shows brightfield images (left) and epifluorescence images(right) according to some embodiments of the present disclosure. C.difficile spores are localised to the upper images and B. subtilisspores are localised to the lower images. Epifluorescence images oflocalised C. difficile spores (upper right) show spots of fluorescencewhich colocalise with C. difficile spores, which is indicative ofbinding of the fluorescently labelled aptamer population selectedagainst CdeC to the C. difficile spores. Spores showing fluorescentsignals are highlighted with circles for clarity. For comparison,epifluorescence images of localised Bacillus subtilis spores (lowerright) are shown. The black box signifies dust on the lens.

FIG. 23 shows brightfield images (left) and epifluorescence images(right) according to some embodiments of the present disclosure. C.difficile spores are localised to the upper images and B. subtilisspores are localised to the lower images. Epifluorescence images oflocalised C. difficile spores (upper right) show spots of fluorescencewhich colocalise with C. difficile spores, which is indicative ofbinding of the fluorescently labelled aptamer population selectedagainst CdeM to the C. difficile spores. Spores showing fluorescentsignals are highlighted with circles for clarity. For comparison,epifluorescence images of localised Bacillus subtilis spores (lowerright) are shown. The black box signifies dust on the lens.

FIG. 24 shows brightfield images (left) and epifluorescence images(right) according to some embodiments of the present disclosure. C.difficile spores are localised to the upper images and B. subtilisspores are localised to the lower images. Epifluorescence images oflocalised C. difficile spores (upper right) shows spots of fluorescencewhich colocalise with C. difficile spores, which is indicative ofbinding of the fluorescently labelled aptamer population selectedagainst CotE to the C. difficile spores. Spores showing fluorescentsignals are highlighted with circles for clarity. For comparison,epifluorescence images of localised Bacillus subtilis spores (lowerright) are shown. The black box signifies dust on the lens.

FIGS. 25A and 25B show brightfield images (left) and epifluorescenceimages (right) according to some embodiments of the present disclosure.C. difficile spores are localised to the upper images and B. subtilisspores are localised to the lower images. Epifluorescence images oflocalised C. difficile spores (upper right) show spots of fluorescencewhich colocalise with C. difficile spores which is indicative of bindingof the fluorescently labelled aptamer population selected against CotECChitinase to the C. difficile spores. Spores showing fluorescent signalsare highlighted with circles for clarity. For comparison,epifluorescence images of localised Bacillus subtilis spores (lowerright) are shown. FIG. 25A illustrates the results of the fluorescentlylabelled aptamer population after 3 rounds of cell selection. FIG. 25Billustrates the results of the fluorescently labelled aptamer populationafter 4 rounds of cell selection. The black box signifies dust on thelens.

FIG. 26 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CotA and the naïve library (Naive) or immobilizedCotA and the monoclonal aptamers: CotA C1 (CotA C1) and CotA B1 (CotAB1) according to some embodiments of the present disclosure.

FIG. 27 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CdeC and the naïve library (Naive) or immobilizedCdeC and the monoclonal aptamer CdeC B3 (CdeC B3) according to someembodiments of the present disclosure.

FIG. 28 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CdeM and the naïve library (Naive) or immobilizedCdeM and the monoclonal aptamer CdeM E2 (CdeM E2) according to someembodiments of the present disclosure.

FIG. 29 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CotE and the naïve library (Naive) or immobilizedCotE and the monoclonal aptamers: CotE D2 (CotE D2) and CotE G1 (CotEG1) according to some embodiments of the present disclosure.

FIG. 30 shows Biolayer Interferometry (BLI) data comparing interactionsbetween immobilized CotEC Chitinase and the naïve library (Naive) orimmobilized CotEC Chitinase and the monoclonal aptamers: Chitinase D11(Chitinase D11), Chitinase D10 (Chitinase D10), and Chitinase H11(Chitinase H11) according to some embodiments of the present disclosure.

FIG. 31A-FIG. 31E show photographs of test samples on a stainless-steelsurface under ambient light conditions without Polilight Flare+2forensic light (505 nm) and without a 590 nm bandpass filter accordingto some embodiments of the present disclosure. For comparison, FIG. 31Aillustrates the untreated stainless-steel surface (negative control 1);FIG. 31B illustrates Clostridium difficile SH11 spores (negative control2); FIG. 31C illustrates horse blood (negative control 3); FIG. 31Dillustrates 10 μm CotE H2 aptamer in buffer (positive control 4); andFIG. 31E illustrates the 10 μm CotE H2 aptamer-Clostridium difficileSH11 spore suspension.

FIG. 32A-FIG. 32E show photographs of test samples on a stainless-steelsurface under ambient light conditions with Polilight Flare+2 forensiclight (505 nm) and without a 590 nm bandpass filter according to someembodiments of the present disclosure. For comparison, FIG. 32Aillustrates the untreated stainless-steel surface (negative control 1);FIG. 32B illustrates Clostridium difficile SH11 spores (negative control2); FIG. 32C illustrates horse blood (negative control 3); FIG. 32Dillustrates 10 μM CotE H2 aptamer in buffer (positive control 4); andFIG. 32E illustrates the 10 μm CotE H2 aptamer-Clostridium difficileSH11 spore suspension.

FIG. 33A-FIG. 33E show photographs of test samples on a stainless-steelsurface under ambient light conditions with Polilight Flare+2 forensiclight (505 nm) and with a 590 nm bandpass filter according to someembodiments of the present disclosure. For comparison, FIG. 33Aillustrates the untreated stainless-steel surface (negative control 1);FIG. 33B illustrates Clostridium difficile SH11 spores (negative control2); FIG. 33C illustrates horse blood (negative control 3); FIG. 33Dillustrates 10 μM CotE H2 aptamer in buffer (positive control 4; andFIG. 33E illustrates the 10 μm CotE H2 aptamer-Clostridium difficileSH11 spore suspension.

FIG. 34A-FIG. 34E show photographs of test samples on a stainless-steelsurface under dark conditions, with exposure to Polilight Flare+2forensic light (505 nm) and with a 590 nm bandpass filter according tosome embodiments of the present disclosure. For comparison, FIG. 34Aillustrates the untreated stainless-steel surface (negative control 1);FIG. 34B illustrates Clostridium difficile SH11 spores (negative control2); FIG. 34C illustrates horse blood (negative control 3); FIG. 34Dillustrates 10 μm CotE H2 aptamer in buffer (positive control 4); andFIG. 34E illustrates the 10 μm CotE H2 aptamer-Clostridium difficileSH11 spore suspension.

FIG. 35A-FIG. 35E show photographs of test samples on a gown surfaceunder ambient light conditions without Polilight Flare+2 forensic light(505 nm) and without 590 nm bandpass filter according to someembodiments of the present disclosure. For comparison, FIG. 35Aillustrates the untreated stainless-steel surface (negative control 1);FIG. 35B illustrates Clostridium difficile SH11 spores (negative control2); FIG. 35C illustrates horse blood (negative control 3); FIG. 35Dillustrates 10 μm CotE H2 aptamer in buffer (positive control 4); andFIG. 35E illustrates the 10 μm CotE H2 aptamer-Clostridium difficileSH11 spore suspension.

FIG. 36A-FIG. 36E show photographs of test samples on a gown surfaceunder ambient light conditions with Polilight Flare+2 forensic light(505 nm) and without 590 nm bandpass filter according to someembodiments of the present disclosure. For comparison, FIG. 36Aillustrates the untreated stainless-steel surface (negative control 1);FIG. 36B illustrates Clostridium difficile SH11 spores (negative control2); FIG. 36C illustrates horse blood (negative control 3); FIG. 36Dillustrates 10 μM CotE H2 aptamer in buffer (positive control 4); andFIG. 36E illustrates the 10 μm CotE H2 aptamer-Clostridium difficileSH11 spore suspension.

FIG. 37A-FIG. 37E show photographs of test samples on a gown surfaceunder ambient light conditions with Polilight Flare+2 forensic light(505 nm) and with 590 nm bandpass filter according to some embodimentsof the present disclosure. For comparison, FIG. 37A illustrates theuntreated stainless-steel surface (negative control 1); FIG. 37Billustrates Clostridium difficile SH11 spores (negative control 2); FIG.37C illustrates horse blood (negative control 3); FIG. 37D illustrates10 μm CotE H2 aptamer in buffer (positive control 4); and FIG. 37Eillustrates the 10 μm CotE H2 aptamer-Clostridium difficile SH11 sporesuspension.

FIG. 38A-FIG. 38E show photographs of test samples on a stainless-steelsurface under dark conditions with Polilight Flare+2 forensic light (505nm) and with 590 nm bandpass filter according to some embodiments of thepresent disclosure. For comparison, FIG. 38A illustrates the untreatedstainless-steel surface (negative control 1); FIG. 38B illustratesClostridium difficile SH11 spores (negative control 2); FIG. 38Cillustrates horse blood (negative control 3); FIG. 38D illustrates 10 μmCotE H2 aptamer in buffer (positive control 4); and FIG. 38E illustratesthe 10 μm CotE H2 aptamer-Clostridium difficile SH11 spore suspension.

DETAILED DESCRIPTION

Clostridium difficile

Clostridium difficile (also referred to as C. difficile) is aGram-positive, anaerobic spore former and is an important nosocomial andcommunity-acquired pathogenic bacterium. C. difficile infections (CDI)are a leading cause of infections worldwide with elevated rates ofmorbidity and mortality. Despite the fact that two major virulencefactors, the enterotoxin TcdA and the cytotoxin TcdB, are essential inthe development of CDI, C. difficile spores are the main vehicle ofinfection, and persistence and transmission of CDI, and are thought toplay an essential role in episodes of CDI recurrence and horizontaltransmission.

Clostridium difficile bacteria are found throughout the environment e.g.in soil, air, water, food products and human and animal faeces. A smallnumber of people carry C. difficile in their intestinal tract withoutshowing any symptoms. However, in other subjects, infection from C.difficile can cause symptoms ranging from diarrhea to life-threateninginflammation of the colon. Complications of C. difficile infection caninclude dehydration, kidney failure, toxic megacolon, perforation of thebowel and even death if the infection is not controlled quickly.

Clostridium difficile bacteria commonly affect older adults in hospitalsor long-term care facilities. Subjects at greater risk of contracting C.difficile include but are not limited to those who have takenantibiotics, those with a compromised immune system, and those who haveundergone abdominal or gastrointestinal surgery. For example, themortality rate of C. difficile infection can be up to 25% in frail,elderly people in hospitals, and it has been postulated that antibiotictherapy disrupts normal gut microbiota, allowing C. difficilecolonization and growth because it is naturally resistant to many drugsused to treat other infections, thereby enabling its toxin production.

An increase of C. difficile infections in subjects previously consideredto be low-risk, for example, younger and otherwise healthy individualswithout exposure to health care facilities, has also been seen in recentyears. A new strain of C. difficile, Type 027, has recently beenidentified, which has been shown to produce more toxins than most othertypes of C. difficile causing a greater proportion of severe disease andapparent higher mortality.

First-line therapy for treating adults with CDI in the U.S. isvancomycin (125 mg, 4 times a day for 10 days) or fidaxomicin (200 mg,twice daily for 10 days) for both severe and non-severe CDI. In the UK,metronidazole (400 mg or 500 mg, 3 times daily for 10-14 days) isconsidered to be the first-line for treating first episodes of mild tomoderate C. difficile infection; and, vancomycin (125 mg 4 times dailyfor 10-14 days) is considered for second episodes or if the infection issevere. An infection is defined as severe when there is a raisedtemperature or white cell count, rising creatinine, or signs or symptomsof severe colitis. Vancomycin may also be used in infections caused bythe type 027 strain. If infection recurs, vancomycin or fidaxomicin (200mg twice daily for 10 days) may be used. In some severe cases, a personmight have to have surgery to remove the infected part of theintestines.

Spores from C. difficile are passed in faeces and can be transmitted tofood, surfaces and objects via unwashed hands. The spores can persistfor weeks or months on surfaces and transmitted via contact with suchsurfaces.

Given the rise in antibiotic resistance and the potential mortalityassociated with C. difficile infection, control measures are of thehighest importance. Current measures include healthcare providers suchas nurses and doctors following protocols including:

-   -   Cleaning hands with soap and water or an alcohol-based hand rub        before and after caring for every patient to prevent C.        difficile and other germs from being passed from one patient to        another on their hands.    -   Carefully cleaning hospital rooms and medical equipment that        have been used for patients with CDI.    -   Giving patients antibiotics only when necessary.    -   Using Contact Precautions to prevent C. difficile from spreading        to other patients. Contact Precautions mean:        -   Whenever possible, keeping patients with C. difficile in a            single room or in a room with another patient who has C.            difficile.        -   Wearing of gloves and a gown over clothing by healthcare            providers while taking care of patients with C. difficile.        -   Wearing of gloves and a gown by visitors.        -   Removing of gloves and gown, and cleaning hands when leaving            the room of a patient with C. difficile.        -   Patients on Contact Precautions are asked to stay in their            hospital rooms as much as possible. They can go to other            areas of the hospital for treatments and tests.

Despite these preventative measures, C. difficile remains a significanthealthcare issue and therefore there is a need for rapid identificationof the presence of C. difficile in an environment in order to minimizeits spread.

Embodiments disclosed herein may at least partially mitigate some of theproblems identified in the prior art.

Embodiments disclosed herein may provide methods and products which haveutility in the detection of C. difficile.

Further features of embodiments of the present invention are describedbelow. The practice of embodiments of the present invention will employ,unless otherwise indicated, conventional techniques of molecularbiology, microbiology, recombinant DNA technology and immunology, knownto one of ordinary skill in the art.

Most general molecular biology, microbiology recombinant DNA technologyand immunological techniques can be found in Sambrook et al, MolecularCloning, A Laboratory Manual (2001) Cold Harbor-Laboratory Press, ColdSpring Harbor, N.Y. or Ausubel et al., Current protocols in molecularbiology (1990) John Wiley and Sons, N.Y. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. For example, the Concise Dictionary of Biomedicineand Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; TheDictionary of Cell and Molecular Biology, 3rd ed., Academic Press; andthe Oxford University Press, provide a person skilled in the art with ageneral dictionary of many of the terms used in this disclosure.

Units, prefixes and symbols are denoted in their Système Internationald′ Unités (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation andnucleic acid sequences are written left to right in 5′ to 3′orientation.

In the following, embodiments are explained in more detail by means ofnon-limiting examples. In the non-limiting, exemplary experiments,standard reagents and buffers free from contamination were used unlessstated otherwise.

Embodiments comprise aptamers capable of specifically binding to C.difficile.

In certain embodiments, the C. difficile is a strain selected from SH11(ribotype RT078), Type 027 and ATCC® 43598. In certain embodiments, theaptamer is capable of binding to a C. difficile spore of strain SH11. Incertain embodiments, the aptamer is capable of binding to a C. difficilespore of strain Type 027. In certain embodiments, the aptamer is capableof binding to a C. difficile spore of strain ATCC® 43598.

Embodiments relate to aptamers which bind to a C. difficile spore.Embodiments comprise an aptamer that binds to a C. difficile spore coatprotein.

C. difficile produces metabolically dormant spores. The spores comprisean outermost exosporium layer which may comprise a number of surfaceproteins. The exosporium layer may comprise one or more proteinsselected from BclA1, BclA2, BclA3, CdeA, CdeB, CdeC and CdeM. Five coatproteins, cotA, cotB, cotCB, cotD, and cotE, were shown to be expressedon the outer coat layers of the spore.

One or more of these proteins may be a target of one or more aptamerherein, and binding to one or more of them by one or more aptamer hereinmay be a basis for a method of detecting C. difficile herein.

In some embodiments, the aptamer specifically binds to a C. difficilespore coat protein as listed in Table 1 below:

TABLE 1 CotA SEQ ID NO: 15 Cot E SEQ ID NO: 16 CotEC SEQ ID NO: 17 CdeCSEQ ID NO: 18 CdeM SEQ ID NO: 19Target Proteins

In an embodiment, the aptamer specifically binds to a target as definedherein. The term “target” as used herein is used to relate to a moleculeselected from at least one of a C. difficile CotA protein, C. difficileCotE protein, C. difficile CdeC protein, C. difficile CdeM protein, C.difficile CotEC chitinase protein, and a C. difficile spore. As usedherein, the terms “target protein” and “target peptide” are usedinterchangeably.

In some embodiments, the aptamer is selected against a whole C.difficile spore. Thus, in some embodiments, the aptamer selectivelybinds to a C. difficile spore.

In some embodiments, the aptamer specifically binds to a surface proteinof the exosporium layer of the C. difficile spore (e.g. CdeC, CdeM). Insome embodiments, the aptamer specifically binds to a coat protein ofthe C. difficile spore (e.g. CotA, CotE, CotEC).

In some embodiments, the target proteins can be naturally occurringtarget proteins or recombinant target proteins listed at Table 2 and maybe a target of one or more aptamers described herein:

TABLE 2 Target Protein SEQ ID NO: CotA 15 Cot E 16 rCotEC 17 CdeC 18CdeM 19 rCotE (LS25) 20CdeC

In some embodiments, the aptamer specifically binds to a C. difficileCdeC protein. The amino acid sequence of CdeC is published underUniProtKB—Q18AS2 (Q18AS2_PEPD6) version 1 and is as set forth in FIG. 1(SEQ ID NO: 18).

In some embodiments, the aptamer binds to an epitope of the CdeC proteinwhich is conserved between C. difficile strains. Thus, in someembodiments, the aptamer is used to detect a plurality of C. difficilestrains in a sample.

CdeM

In some embodiments, the aptamer selectively binds to an amino acidsequence of a C. difficile surface-bound CdeM protein. CdeM is acysteine rich protein which is understood to be required for themorphogenesis of the coat and exosporium layer of spores. An amino acidsequence of a C. difficile protein is published underUniProtKB—A0A3T1GTU1 (A0A3T1GTU1_CLODI) (version 1) and shown in FIG. 2(SEQ ID NO: 19).

In some embodiments, the aptamer binds to an epitope of the CdeM proteinwhich is conserved between C. difficile strains. Thus, in someembodiments, the aptamer is used to detect a plurality of C. difficilestrains in a sample.

In some embodiments, the spores comprise a spore coat. The spore coatmay comprise a plurality of proteins including, but not limited to CotAand CotB for example.

CotA

In some embodiments, the aptamer specifically binds to a protein encodedby a C. difficile CotA gene. The protein may be referred to herein aseither CotA or “spore coat assembly protein”.

An amino acid sequence of CotA is published under UniProtKB AccessionNo. Q186G8 (Q186G8_PEPD6) version 1 and shown in FIG. 3 (SEQ ID NO: 15).

CotE and CotEC Chitinase

In some embodiments, the aptamer specifically binds to a C. difficileprotein encoded by a CotE gene. An amino acid sequence of a CotE protein(also referred to as peroxiredoxin) is published under accession numberUniProtKB—Q18BV5 (Q18BV5_PEPD6) and is shown in FIG. 4 (SEQ ID NO: 16).

In some embodiments, aptamers were raised to a recombinant form of CotEreferred to as “rCotE” (also referred to as LS25). The amino acidsequence of rCotE is shown in FIG. 5A and consists of amino acidresidues N281-F712 (SEQ ID NO: 20). The recombinant protein comprises achitinase domain and a sequence unique to CotE, as shown in FIG. 5A.

In some embodiments, the aptamer specifically binds to a recombinant C.difficile protein referred to as “rCotEC” (also referred to as AB45).The amino acid sequence of rCotEC is shown in FIG. 5B and consists ofamino acid residues N381-F712 (SEQ ID NO: 17).

In some embodiments, the aptamers are selected against a tagged rCotECprotein, including but not limited to His-tagged rCotEC protein.

In some embodiments, the aptamers are selected against a taggedrecombinant C. difficile protein including but not limited to His-taggedC. difficile protein. Other protein tags commonly used in the art toassist with protein purification may be used as well.

In some embodiments, the aptamer is selected against a whole C.difficile spore. Thus, in some embodiments, the aptamer selectivelybinds to a C. difficile spore.

In an embodiment, the aptamer specifically binds to an epitope in a C.difficile CotA protein.

In an embodiment, the aptamer specifically binds to an epitope in a C.difficile CotE protein.

In an embodiment, the aptamer specifically binds to an epitope in a C.difficile CdeC protein.

In an embodiment, the aptamer specifically binds to an epitope in a C.difficile CdeM protein.

In an embodiment, the aptamer specifically binds to an epitope in a C.difficile CotEC chitinase protein.

An aptamer binds “specifically” to a target as defined herein if theaptamer binds with preferential or high affinity to the target proteinbut does not bind or binds with only low affinity to other structurallyrelated molecules (e.g. Bacillus subtilis spores.) In some embodiments,the dissociation constant for the target protein is in the micro-molarrange. In some embodiments, the dissociation constant for the targetprotein is in the nano-molar range. In some embodiments, thedissociation constant for the target protein is in the pico-molar range.In some embodiments, the dissociation constant is about 0.1 nM or less.In some embodiments, the dissociation constant is about 0.1 nM to about1 nM. In some embodiments, the dissociation constant is about 1 nM toabout 10 nM. In some embodiments, the dissociation constant is about 10nM to about 100 nM. In some embodiments, the dissociation constant isabout 100 nM to about 1000 nM. Lower affinity binding may refer tobinding that occurs at less affinity than to a target protein. The loweraffinity binding may be selected from the range of less than 1 fold to 2fold, less than 2 fold to 5 fold, less than 5 fold to 10 fold, less than10 fold to 50 fold, less than 50 fold to 100 fold, less than 100 fold to1000 fold, less than 1000 fold to 10000 fold, or less than 10000 fold to100000 fold of binding to the target protein.

Aptamers

The aptamers described herein are small artificial ligands, compromisingDNA, RNA or modifications thereof, capable of specifically binding to atarget as defined herein with high affinity and specificity.

As used herein, “aptamer,” “nucleic acid molecule,” or “oligonucleotide”are used interchangeably to refer to a non-naturally occurring nucleicacid molecule that has a desirable action on a target as defined herein.

In some embodiments, the aptamers may be DNA aptamers. For example, theaptamers may be formed from single-stranded DNA (ssDNA). In someembodiments, the aptamers may be RNA aptamers. For example, the aptamerscan be formed from single-stranded RNA (ssRNA).

In some embodiments, there is provided an aptamer comprising a nucleicacid sequence selected from a nucleic acid sequence as set forth inTable 3.

TABLE 3 Aptamer Sequences Sequence

TCTTACGATCCTCACCTGCTAGCACA CCCATATCCCATGC

(SEQ ID NO: 1)

GGGTTGCGACATGGTGGTAAGAGCTC AGCCCGTTCCCATA

(SEQ ID NO: 2)

ACGGCCTGTTCGTAAGACCCTTACAG ACTAGTTTTTCCCT

(SEQ ID NO: 3)

CCTATTAGCTGTATCGATCCGTTTAG TCGCTCCTCCGATA

(SEQ ID NO: 4)

CTGGTAAATCGATGACCGCTGCCTCG CCTGAGTAATCATC

(SEQ ID NO: 5)

CGTGGACTGGTCGGGTTTGGATTCG GCAGATGAATCAGTA

(SEQ ID NO: 6)

CTTGTAAGAAGAACAATCGCCGCTT CGCCTGAATAGGTTC

(SEQ ID NO: 7)

GGACCGTTGCCTCGCCCGAGTAATC CGCCATCGCCTTTCC

(SEQ ID NO: 8)

TTAAGTTCTGGGGACACGTGATGAAC GCATTTAATGGGGC

(SEQ ID NO: 9)

CGTGGACTGGTCGGGTTTGGATCGGC AGATGAATCACTA

(SEQ ID NO: 10)

GGCTGTGTGACTTGACCTTTGGAATG GGTGGGAGGGATGG

(SEQ ID NO: 11)

GGTGTGGTGACCTTGACCTATGGAAC CTGGTTGTA

(SEQ ID NO: 12)

TCGACATTTCCGCCCCGACGGCCCTC CTAGTGATGGGGAGA

(SEQ ID NO: 13)

CTTCCATTCACCTACCGAGCTAAGCG TTCGACTTAGGTCT

(SEQ ID NO: 14) ATCGATGACCGCTGCCTCGCCTGAGTAATCATC

(SEQ ID NO: 23) CCATACTCAATGCTCTTACGATCCTCATCAACC (SEQ ID NO: 24)CCAGTGTAGACTACTCAATGCTCTTACGATCCTCATCAACC (SEQ ID NO: 25)AGTGTAGACTACTCAATGCGGCTGGCCACAGGTCAACC (SEQ ID NO: 26)

Primer Regions are Indicated in Bold and Italic:

TABLE 4 ID Sequence Target C.diff_F1

TCTTACGATCCTCACCTGCTAGCACACCCA C.diff spores TATCCCATGC

 (SEQ ID NO: 1) C.diff_G1

GGGTTGCGACATGGTGGTAAGAGCTCAGCC C.diff spores CGTTCCCATA

 (SEQ ID NO: 2) C.diff_E2

ACGGCCTGTTCGTAAGACCCTTACAGACTA C.diff spores GTTTTTCCCT

 (SEQ ID NO: 3) Chitinase_D10

CCTATTAGCTGTATCGATCCGTTTAGTCGC CotEC TCCTCCGATA

 (SEQ ID NO: 4) Chitinase Chitinase_D11

CTGGTAAATCGATGACCGCTGCCTCGCCTG CotEC AGTAATCATC

 (SEQ ID NO: 5) Chitinase CdeC_D1

CGTGGACTGGTCGGGTTTGGATTCGGCAGA CdeC TGAATCAGTA

 (SEQ ID NO: 6) Chitinase_H11

CTTGTAAGAAGAACAATCGCCGCTTCGCCT CotEC GAATAGGTTC

 (SEQ ID NO: 7) Chitinase Chitinase_D7

GGACCGTTGCCTCGCCCGAGTAATCCGCCA CotEC TCGCCTTTCC

 (SEQ ID NO: 8) Chitinase CotA_B1

TTAAGTTCTGGGGACACGTGATGAACGCAT CotA TTAATGGGGC

 (SEQ ID NO: 9) CotA_C1

CGTGGACTGGTCGGGTTTGGATTCGGCAGA CotA TGAATCACTA

 (SEQ ID NO: 10) CotE_H2

GGCTGTGTGACTTGACCTTTGGAATGGGTG CotE GGAGGGATGG

 (SEQ ID NO: 11) CotE_E2

GGTGTGGTGACCTTGACCTATGGAACCTGG CotE TTGTA

 (SEQ ID NO: 12) CotE_D2

TCGACATTTCCGCCCCGACGGCCCTCCTAG CotE TGATGGGGAGA

 (SEQ ID NO: 13) CdeM_E2

CTTCCATTCACCTACCGAGCTAAGCGTTCG CdeM ACTTAGGTCT

 (SEQ ID NO: 14) Chitinase_D11 ATCGATGACCGCTGCCTCGCCTGAGTAATCATC

CotEC (SEQ ID NO: 23) Chitinase C.diff_F1CCATACTCAATGCTCTTACGATCCTCATCAACC (SEQ ID NO: 24) C.diff sporesC.diff_G1 CCAGTGTAGACTACTCAATGCTCTTACGATCCTCATCAACC C.diff spores(SEQ ID NO: 25) CotE_H2 AGTGTAGACTACTCAATGCGGCTGGCCACAGGTCAACC CotE(SEQ ID NO: 26)

In some embodiments, the aptamers are RNA aptamers and comprise asequence in which one or some or all of the deoxyribonucleotides in anyof the sequences set forth in SEQ ID NO. 1 to 14 and SEQ ID NO: 23 to 26are substituted for their equivalent ribonucleotide residues AMP, GMP,UMP or CMP.

The aptamers of embodiments of the invention may comprise modifiednucleic acids as described herein.

In some embodiments, the aptamers of the invention are prepared usingprinciples of in vitro selection known in the art, that includeiterative cycles of target binding, partitioning and preferentialamplification of target binding sequences. Selection may be performedusing immobilized target proteins. Immobilization may include, but isnot limited to, immobilization to a solid surface. In a non-limitingexample, the solid surface may be beads. In a non-limiting example, thesolid surface may be magnetic beads.

Non-limiting examples of amplification methods include polymerase chainreaction (PCR), ligation amplification (or ligase chain reaction, LCR),strand displacement amplification, nucleic acid sequence-basedamplification, and amplification methods based on the use of Q-betareplicase. In a non-limiting embodiment, at least one type of aptamermay be immobilized on a solid surface during amplification. Each ofthese exemplary methods is well known in the art.

In some embodiments, the aptamers are selected from a nucleic acidmolecule library such as a single-stranded DNA or RNA nucleic acidmolecule library. The aptamers may be selected from a “universal aptamerselection library” that is designed such that any selected aptamers needlittle to no adaptation to convert into any of the listed assay formats.

Once selected, the aptamer may be further modified before being usede.g. to remove one or both primer sequences and/or parts of therandomised sequence not required for target binding.

Typically, aptamers of the embodiments of the invention comprise a firstprimer region (e.g. at the 5′ end), a second primer region (e.g. at the3′ end), or both. The primer regions may serve as primer binding sitesfor PCR amplification of the library and selected aptamers.

The skilled person would understand different primer sequences can beselected depending, for example, on the starting library and/or aptamerselection protocol. In some embodiments, the primer comprises orconsists of a nucleic acid sequence of SEQ ID NO: 21 and/or 22. In anembodiment, aptamers may comprise SEQ ID NO: 21 and/or 22. In otherembodiments, any one of one to all of the nucleotides disclosed by SEQID NO: 21 or 22 may be modified. The primer region length may also bevaried.

In some embodiments, the primers are shown in Table 5

TABLE 5 CCAGTGTAGACTACTCAATGC (primer) SEQ ID NO: 21GTACTATCCACAGGTCAACC (primer) SEQ ID NO: 22

The first primer region and/or second region may comprise a detectablelabel as described herein. As used herein the terms “detectable label”and “detectable moiety” are used interchangeably. In an embodiment, thefirst and/or second primer region may be fluorescently labelled.Non-limiting examples of fluorescent labels include but are not limitedto fluorescein, green fluorescent protein (GFP), yellow fluorescentprotein, cyan fluorescent protein, and others. In an embodiment, afluorescein label is used. In some embodiments, other forms of detectingthe primer may be used, including but not limited to phosphate (PO₄)labelling, isotope labelling, electrochemical sensors, colorimetricbiosensors, and others.

In some embodiments, the aptamers of the invention comprise or consistof a nucleic acid sequence selected from any one of SEQ ID NOs: 1 to 14.

In some embodiments, aptamers of the invention comprise or consist of anucleic acid sequence having at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or more sequenceidentity to the nucleotide sequence of any one of SEQ ID NOs: 1 to 14 orSEQ ID NOs: 23 to 26.

As used herein, “sequence identity” refers to the percentage ofnucleotides in a candidate sequence that are identical with thenucleotides in said sequences after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent nucleic acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, CLUSTALW or Megalign (DNASTAR)software. For example, % nucleic acid sequence identity values can begenerated using sequence comparison computer programs found on theEuropean Bioinformatics Institute website (www.ebi.ac.uk).

As used herein, when describing the percent identity of a nucleic acid,such as an aptamer, the sequence of which is at least, for example,about 90% identical to a reference nucleotide sequence, it is intendedthat the nucleic acid sequence is identical to the reference sequenceexcept that the nucleic acid sequence may include up to ten pointmutations (e.g. substitution, deletion, insertion) per each 100nucleotides of the reference nucleic acid sequence. These mutations mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those 5′ or 3′ terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a minimal effective fragment of SEQ ID NOs: 1 to 14 or SEQ IDNOs: 23-26. Herein, a “minimal effective fragment” is understood to meana fragment (e.g. portion) of the full-length aptamer capable of bindingto a target as defined herewith with the same or improved affinity ascompared to the full-length aptamer. A minimal effective fragment maycompete for binding to a target as defined herein with the full-lengthaptamer.

In some embodiments, the aptamers comprise, consist essentially of, orconsist of at least 20 contiguous nucleic acid residues of any of thesequences as set forth in any one of SEQ ID NOs: 1 to 14 or SEQ ID NOs:23-26 and show equivalent or improved binding to the target molecule. Insome embodiments, the aptamers of the invention comprise, consistessentially of, or consist of at least 20 contiguous nucleic acidresidues of any of the sequences as set forth in any one of SEQ ID NOs:1 to 14 or SEQ ID NOs: 23-26 and show adequate binding to the targetmolecule. Adequate binding includes binding to target molecule thatoccurs with affinity and specificity as described herein, or an affinityand/or specificity of binding less than that of the full-length aptamersequence above but still capable of delivering a report of the presenceof its respective target.

In some embodiments, an aptamer of the invention comprises, consistsessentially of, or consists of at least 25 contiguous nucleotides of anyof the sequences as set forth in any one of SEQ ID NOs: 1 to 14 or SEQID NOs: 23-26.

In some embodiments, an aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 1. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 1, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 2. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 2, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 3. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 3, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 4. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 4, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 5. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 5, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 6. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 6, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 7. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 7, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 8. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 8, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 9. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 9, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 10. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 10, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 11. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 11, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75contiguous nucleotides in the nucleic acid sequence of SEQ ID NO: 12.The aptamer may comprise, consist essentially of, or consist of any spanof contiguous nucleotides from SEQ ID NO: 12, where the span has alength chosen in one nucleotide increments from 25 nucleotides to fulllength.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, or 81 nucleotides in the nucleic acid sequence of SEQ IDNO: 13. The aptamer may comprise, consist essentially of, or consist ofany span of contiguous nucleotides from SEQ ID NO: 13, where the spanhas a length chosen in one nucleotide increments from 25 nucleotides tofull length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 contiguous nucleotides in the nucleic acid sequence ofSEQ ID NO: 14. The aptamer may comprise, consist essentially of, orconsist of any span of contiguous nucleotides from SEQ ID NO: 14, wherethe span has a length chosen in one nucleotide increments from 25nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguousnucleotides in the nucleic acid sequence of SEQ ID NO: 23. The aptamermay comprise, consist essentially of, or consist of any span ofcontiguous nucleotides from SEQ ID NO: 23, where the span has a lengthchosen in one nucleotide increments from 25 nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32 or 33 contiguous nucleotidesin the nucleic acid sequence of SEQ ID NO: 24. The aptamer may comprise,consist essentially of, or consist of any span of contiguous nucleotidesfrom SEQ ID NO: 24, where the span has a length chosen in one nucleotideincrements from 25 nucleotides to full length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, or 41 contiguous nucleotides in the nucleic acid sequence of SEQ IDNO: 25. The aptamer may comprise, consist essentially of, or consist ofany span of contiguous nucleotides from SEQ ID NO: 25, where the spanhas a length chosen in one nucleotide increments from 25 nucleotides tofull length.

In some embodiments, the aptamer comprises, consists essentially of, orconsists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38contiguous nucleotides in the nucleic acid sequence of SEQ ID NO: 26.The aptamer may comprise, consist essentially of, or consist of any spanof contiguous nucleotides from SEQ ID NO: 26, where the span has alength chosen in one nucleotide increments from 25 nucleotides to fulllength.

In some embodiments, these sequences relate to aptamer fragments withequivalent, suitable, or improved binding to a target protein asdescribed herein as compared to full-length aptamer.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 51, 52, 53, 54, 55, 60 or more consecutive nucleotides of asequence having at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or more identity with any ofSEQ ID NOs: 1 to 14 or SEQ ID NOs: 23-26. In this context the term“about” typically means the referenced nucleotide sequence length plusor minus 10% of that referenced length.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence having at least 85% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 85% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 85% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 85% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 85% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence having at least 90% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 90% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 90% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 90% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 90% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence having at least 95% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 95% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 95% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 95% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 95% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence having at least 96% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 96% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 96% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 96% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 96% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85 or more consecutive nucleotides of asequence having at least 97% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 97% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 97% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 97% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 97% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence having at least 98% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 98% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 98% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 98% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 98% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence having at least 99% or more identity with any of SEQ ID NOs: 1to 14. In some embodiments, aptamers comprise, consist essentially of,or consist of a nucleic acid sequence comprising at least about 25, 30,35, or more consecutive nucleotides of a sequence having at least 99% ormore identity with SEQ ID NO: 23. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 25, 30, or more consecutive nucleotides of asequence having at least 99% or more identity with SEQ ID NO: 24. Insome embodiments, aptamers comprise, consist essentially of, or consistof a nucleic acid sequence comprising at least about 25, 30, 35, 40, ormore consecutive nucleotides of a sequence having at least 99% or moreidentity with SEQ ID NO: 25. In some embodiments, aptamers comprise,consist essentially of, or consist of a nucleic acid sequence comprisingat least about 25, 30, 35, or more consecutive nucleotides of a sequencehaving at least 99% or more identity with SEQ ID NO: 26.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of asequence comprising any one of SEQ ID NOs: 1 to 14.

In some embodiments, aptamers comprise, consist essentially of, orconsist of a nucleic acid sequence comprising at least about 30 or moreconsecutive nucleotides of a sequence comprising SEQ ID NO: 23. In someembodiments, aptamers comprise, consist essentially of, or consist of anucleic acid sequence comprising at least about 30 or more consecutivenucleotides of a sequence comprising SEQ ID NO: 24. In some embodiments,aptamers comprise, consist essentially of, or consist of a nucleic acidsequence comprising at least about 30 or more consecutive nucleotides ofa sequence comprising SEQ ID NO: 25. In some embodiments, aptamerscomprise, consist essentially of, or consist of a nucleic acid sequencecomprising at least about 30 or more consecutive nucleotides of asequence comprising SEQ ID NO: 26.

The aptamers may comprise natural or non-natural nucleotides and/or basederivatives (or combinations thereof). In some embodiments, the aptamerscomprise one or more modifications such that they comprise a chemicalstructure other than deoxyribose, ribose, phosphate, adenine (A),guanine (G), cytosine (C), thymine (T), or uracil (U). The aptamers maybe modified at the nucleobase, at the sugar or at the phosphatebackbone.

In some embodiments, the aptamers comprise one or more modifiednucleotides. Exemplary modifications include for example nucleotidescomprising an alkylation, arylation or acetylation, alkoxylation,halogenation, amino group, or another functional group. Examples ofmodified nucleotides include, but are not limited to, 2′-fluororibonucleotides, 2′-NH₂—, 2′-OCH₃— and 2′-O-methoxyethylribonucleotides, which are used for RNA aptamers.

The aptamers may be wholly or partly phosphorothioate or DNA,phosphorodithioate or DNA, phosphoroselenoate or DNA,phosphorodiselenoate or DNA, locked nucleic acid (LNA), peptide nucleicacid (PNA), N3′-P5 ‘phosphoramidate RNA/DNA, cyclohexene nucleic acid(CeNA), tricyclo DNA (tcDNA) or spiegelmer, or the phosphoramidatemorpholine (PMO) components or any other modification known to thoseskilled in the art (see also Chan et al., Clinical and ExperimentalPharmacology and Physiology (2006) 33, 533-540).

Some of the modifications may allow the aptamers to be stabilizedagainst nucleic acid-cleaving enzymes. In the stabilization of theaptamers, a distinction can generally be made between the subsequentmodification of the aptamers and the selection with already modifiedRNA/DNA. The stabilization may not affect the affinity of the modifiedRNA/DNA aptamers but may prevent the rapid decomposition of the aptamersin an organism, biological solutions, or solutions, by RNases/DNases. Anaptamer is referred to as stabilized if the half-life of the aptamer inthe sample (e.g. biological medium, organism, solution) is greater thanone minute, greater than one hour, or greater than one day. The aptamersmay be modified with reporter molecules, which may enable detection ofthe labelled aptamers. Reporter molecules may also contribute toincreased stability of the aptamers.

Aptamers form a three-dimensional structure that depends on theirnucleic acid sequence. The three-dimensional structure of an aptamer mayarise due to Watson and Crick intramolecular base pairing, Hoogsteenbase pairing (quadruplex), wobble-pair formation, or other non-canonicalbase interactions. In some embodiments, the three-dimensional structureenables aptamers, analogous to antigen-antibody binding, to bind targetstructures accurately. A nucleic acid sequence of an aptamer may, underdefined conditions, have a three-dimensional structure that is specificto a defined target structure.

Embodiments comprise competitive aptamers that compete for binding to atarget protein as defined herein with aptamers as described herein.Embodiments comprise competitive aptamers that compete for binding to atarget protein as defined herein with the aptamers set forth in any oneof SEQ ID NOs: 1 to 14 or SEQ ID NOs: 23-26, or with aptamers having anucleic acid sequence having at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% sequence identityto the nucleotide sequence of any one of SEQ ID NOs: 1 to 14 or SEQ IDNOs: 23-26. Embodiments comprise competitive one or more aptamers thatcompete for binding to a target protein as defined herein with one ormore of the aptamers described above. In some embodiments, competitionassays may be used identify a competitive aptamer that competes forbinding to a target protein as defined herein. In an exemplary,non-limiting, competition assay, an immobilized target protein asdefined herein is incubated in a solution comprising a first labelledaptamer that binds to a target protein as defined herein and a secondunlabelled aptamer that is being tested for its ability to compete withthe first aptamer for binding to a target protein as defined herein. Asa control, an immobilized target protein as defined herein may beincubated in a solution comprising the first labelled aptamer but notthe second unlabelled aptamer. After incubation under conditionspermissive for binding of the first aptamer to a target protein asdefined herein excess unbound aptamer may be removed, and the amount oflabel associated with immobilized target protein as defined hereinmeasured. If the amount of label associated with immobilized target asdefined herein is substantially reduced in the test sample relative tothe control sample, then that indicates that the second aptamer iscompeting with the first aptamer for binding to a target protein asdefined herein.

Support

In some embodiments, the target peptide or protein is attached to asupport. In a non-limiting example, the support may be a solid support.Non-limiting examples of a solid support include a membrane or a bead.In some embodiments, the support may be a two-dimensional support. Anon-limiting example of a two-dimensional support is a microplate. Insome embodiments, the support may be a three-dimensional support. Anon-limiting example of a three-dimensional support is a bead. In someembodiments, the support may comprise at least one magnetic bead.

In some embodiments, the protein comprises a polyhistidine tag (His tag)tag (e.g. hexa-histidine tag) at its N- or C-termini. For example, theprotein can be a recombinant protein having Histidine residues at itsC-terminus or its N-terminus. In some embodiments, the His-taggedprotein can be immobilized onto a support carrying a histidine bindingagent. For example, the His-tagged protein can be immobilized to asupport having nickel nitrilotriacetic acid (Ni-NTA).

In some embodiments, the support may comprise at least one nanoparticle.A non-limiting example of a nanoparticle is a gold nanoparticle or thelike. In yet further embodiments, the support may comprise a microtiteror other assay plate, a strip, a membrane, a film, a gel, a chip, amicroparticle, a nanofiber, a nanotube, a micelle, a micropore, ananopore, or a biosensor surface. In some embodiments, the biosensorsurface may be a probe tip surface, a biosensor flow-channel, orsimilar.

In some embodiments, the support comprises a membrane. Non-limitingexamples of a membrane include a nitrocellulose, a polyethylene (PE), apolytetrafluoroethylene (PTFE), a polypropylene (PP), a celluloseacetate (CA), a polyacrylonitrile (PAN), a polyimide (PI), a polysulfone(PS), a polyethersulfone (PES) membrane or an inorganic membranecomprising aluminum oxide (Al₂O₃), silicon oxide (SiO₂), and/orzirconium oxide (ZrO₂). Non-limiting examples of materials from which asupport may be made include inorganic polymers, organic polymers,glasses, organic and inorganic crystals, minerals, oxides, ceramics,metals, especially precious metals, carbon, and semiconductors. In anembodiment, the organic polymer is a polymer based on polystyrene.Biopolymers, including but not limited to cellulose, dextran, agar,agarose and Sephadex, which may be functionalized in particular asnitrocellulose or cyanogen bromide Sephadex, may be polymers in asupport.

Detectable Labels

In some embodiments, the aptamers of the invention are used to detectand/or quantify the amount of a target as defined herein in a sample.Typically, the aptamers comprise a detectable label. Any label capableof facilitating detection and/or quantification of the aptamers may beused herein. Non-limiting examples of detectable labels are describedbelow.

In some embodiments, the detectable label is a fluorescent moiety, e.g.a fluorescent compound. In some embodiments, the aptamer comprises afluorescent and a quencher compound. Fluorescent and quencher compoundsare known in the art. See, for example, Mary Katherine Johansson,Methods in Molecular Biol. 335: Fluorescent Energy Transfer Nucleic AcidProbes: Designs and Protocols, 2006, Didenko, ed., Humana Press, Totowa,N.J., and Marras et al., 2002, Nucl. Acids Res. 30, e122 (incorporatedby reference herein).

In some embodiments, the detectable label is FAM. In some embodiments,the FAM-label is conjugated to the 5′ end or the 3′ end of the aptamer.One of ordinary skill in the art would understand that the label may belocated at any suitable position within the aptamer.

In some embodiments, the aptamer comprises a FAM fluorophore at its 5′end. In some embodiments, the aptamer is synthesized by incorporatingphosphoramidite one at a time into the nucleic acid chain and theFAM-labeled phosphoramidite is incorporated through the synthesisprocess. In some embodiments, the FAM fluorophore is attached at the 5′end of the aptamer via a linker. In some embodiments, the detectablelabel is attached to an aptamer described herein via a moiety selectedfrom a thiol group, an amine group, an azide, six-carbon linker, and anaminoallyl group and combinations thereof. In some embodiments, the FAMlabel can be incorporated into the aptamer using a forward primer with aFAM on the 5′ end. In some embodiments, the aptamer can be prepared bysolid phase synthesis with the FAM label already in place, attached tothe 5′ end as in the primer.

Moieties that result in an increase in detectable signal when inproximity of each other may also be used herein, for example, as aresult of fluorescence resonance energy transfer (“FRET”); suitablepairs include but are not limited to fluorescein andtetramethylrhodamine; rhodamine 6G and malachite green, and FITC andthiosemicarbazole, to name a few.

In some embodiments, the detectable label is and/or comprises a moietyselected from at least one of the following non-limiting examples: afluorophore, a nanoparticle, a quantum dot, an enzyme, a radioactiveisotope, a pre-defined sequence portion, a biotin, a desthiobiotin, athiol group, an amine group, an azide, an aminoallyl group, adigoxigenin, an antibody, a catalyst, a colloidal metallic particle, acolloidal non-metallic particle, an organic polymer, a latex particle, ananofiber, a nanotube, a dendrimer, a protein, and a liposome.

In some embodiments, the detectable label is a fluorescent protein suchas Green Fluorescent Protein (GFP) or any other fluorescent proteinknown to those skilled in the art.

In some embodiments, the detectable label is an enzyme. For example, theenzyme may be selected from horseradish peroxidase, alkalinephosphatase, urease, β-galactosidase or any other enzyme known to thoseskilled in the art.

In some embodiments, the nature of the detection will be dependent onthe detectable label used. For example, the label may be detectable byvirtue of its colour e.g. gold nanoparticles. A colour can be detectedquantitatively by an optical reader or camera e.g. a camera with imagingsoftware.

In some embodiments, the detectable label is a fluorescent label e.g. aquantum dot. In such embodiments, the detection means may comprise afluorescent plate reader, strip reader or similar, which is configuredto record fluorescence intensity.

In some embodiments in which the detectable label is an enzyme label,non-limiting detection means may, for example, be colorimetric,chemiluminescence and/or electrochemical (including, but not limited tousing an electrochemical detector). Electrochemical sensing may bethrough conjugation of a redox reporter (including, but not limited tomethylene blue or ferrocene) to one end of the aptamer and a sensorsurface to the other end. A change in aptamer conformation upon targetbinding may change the distance between the reporter and sensor toprovide a readout.

In some embodiments, the detectable label may further comprise enzymes,including but not limited to, horseradish peroxidase (HRP), Alkalinephosphatase (APP) or similar, to catalytically turnover a substrate togive an amplified signal.

Embodiments comprise a complex (e.g. conjugate) comprising aptamers ofthe invention and a detectable molecule. Typically, the aptamers of theinvention are covalently or physically conjugated to a detectablemolecule.

In some embodiments, the detectable molecule is a visual, optical,photonic, electronic, acoustic, opto-acoustic, mass, electrochemical,electro-optical, spectrometric, enzymatic, or otherwise physically,chemically or biochemically detectable label.

In some embodiments, the detectable molecule is detected byluminescence, UV/VIS spectroscopy, enzymatically, electrochemically orradioactively. Luminescence refers to the emission of light. Forexample, photoluminescence, chemiluminescence and bioluminescence areused for detection of the label. In photoluminescence or fluorescence,excitation occurs by absorption of photons. Exemplary fluorophoresinclude, but are not limited to, bisbenzimidazole, fluorescein, acridineorange, Cy5, Cy3 or propidium iodide, which can be covalently coupled toaptamers, tetramethyl-6-carboxyhodamine (TAMRA), Texas Red (TR),rhodamine, Alexa Fluor dyes (et al. Fluorescent dyes of differentwavelengths from different companies).

In some embodiments, the detectable molecule is a colloidal metallicparticle, including but not limited to a gold nanoparticle, colloidalnon-metallic particle, quantum dot, organic polymer, latex particle,nanofiber (carbon nanofiber, as a non-limiting example), nanotube(carbon nanotube, as a non-limiting example), dendrimer, protein orliposome with signal-generating substances. Colloidal particles may bedetected colorimetrically.

In some embodiments, the detectable molecule is an enzyme. In someembodiments, the enzyme may convert substrates to coloured products.Examples of the enzyme include but are not limited toperoxidase,luciferase, β-galactosidase or alkaline phosphatase. For example, thecolourless substrate X-gal is converted by the activity ofβ-galactosidase to a blue product whose colour is visually detected.

In some embodiments, the detection molecule is a radioactive isotope.The detection may also be carried out by means of radioactive isotopeswith which the aptamer is labelled, including but not limited to ³H,¹⁴C, ³²P, ³³P, ³⁵S or ¹²⁵I. In an embodiment, scintillation counting maybe conducted, and thereby the radioactive radiation emitted by theradioactively labelled aptamer target complex is measured indirectly. Ascintillator substance is excited by the isotope's radioactiveemissions. During the transition of the scintillation material, back tothe ground state, the excitation energy is released again as flashes oflight, which are amplified and counted by a photomultiplier.

In some embodiments, the detectable molecule is selected fromdigoxigenin and biotin. Thus, the aptamers may also be labelled withdigoxigenin or biotin, which are bound for example by antibodies orstreptavidin, which may in turn carry a label, such as an enzymeconjugate. The prior covalent linkage (conjugation) of an aptamer withan enzyme can be accomplished in several known ways. Detection ofaptamer binding may also be achieved through labelling of the aptamerwith a radioisotope in an RIA (radioactive immunoassay), preferably with¹²⁵I, or by fluorescence in a FIA (fluoroimmunoassay) with fluorophores,preferably with fluorescein or fluorescein isothiocyanate (FITC).

Embodiments comprise methods for detecting the presence, absence oramount of a target as defined herein in a sample. In the methods, thesample may be interacted (i.e. contacted) with an aptamer as describedherein. For example, the sample and aptamers as described herein may beincubated under conditions sufficient for at least a portion of theaptamer to bind to a target as defined herein in the sample.

A person skilled in the art will understand that the conditions requiredfor binding to occur between the aptamers described herein and a targetas defined herein. In some embodiments, the sample and aptamer may beincubated at temperatures between about 4° C. and about 40° C. In someembodiments, the sample and aptamer may be incubated at temperaturesbetween about 20° C. and about 37° C. In some embodiments, the sampleand aptamer may be incubated at or about 22° C. The incubationtemperature may be selected from the range of 4° C. to less than 20° C.,20° C. to less than 22° C., 22° C. to less than 24° C., 24° C. to lessthan 26° C., 26° C. to less than 28° C., 28° C. to less than 30° C., 30°C. to less than 32° C., 32° C. to less than 34° C., 34° C. to less than36° C., 36° C. to 37° C., and 37° C. to 40° C. In some embodiments, thesample and aptamer may be diluted to different concentrations (e.g. atleast about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% 80% v/v ormore) with a buffer (exemplary buffers include but are not limited toPBS). The diluted concentrations may be selected from the range of 1% toless than 5%, 5% to less than 10%, 10% to less than 20%, 20% to lessthan 30%, 30% to less than 40%, 40% to less than 50%, 50% to less than60%, 60% to less than 70%, 70% to less than 80%, or 80% to less than90%. In some embodiments, the aptamer concentration before dilution maybe from 100 nM to 50 μM. In some embodiments, the aptamer concentrationbefore dilution may be selected from the range of 100 nM to 500 nM, 500nM to 1 μM, 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, 10 μM to 15 μM,15 μM to 20 μM, 20 μM to 30 μM, 30 μM to 40 μM, 40 μM to 50 μM, 50 μM to60 μM, 60 μM to 70 μM, 70 μM to 80 μM, 80 μM to 90 μM, 90 μM to 100 μM.In some embodiments, the aptamer concentration before dilution may be aconcentration selected from the ranges described herein in. The selectedvalue may be selected from 0.1 μM increment concentrations in a rangeherein. In some embodiments, the aptamer concentration before dilutionmay be 2 μM. In some embodiments, the sample and aptamer may beincubated whilst shaking and/or mixing. In some embodiments, the sampleand aptamer are incubated for at least 1 minute, at least 5 minutes, atleast 15 minutes, at least 1 hour, or more. The sample and aptamer maybe incubated for 1 minute to less than 5 minutes, 5 minutes to less than15 minutes, 15 minutes to less than one hour, one hour to less than 24hours, 24 hours to less than 48 hours.

In some embodiments, binding of the aptamer and a target as definedleads to formation of an aptamer-target complex. The binding or bindingevent may be detected, for example, visually, optically, photonically,electronically, acoustically, opto-acoustically, by mass,electrochemically, electro-optically, spectrometrically, enzymaticallyor otherwise chemically, biochemically or physically as describedherein.

The binding of aptamer and the target may be detected using any suitabletechnique. As discussed above, for example, binding of the aptamer andthe target may be detected using a biosensor. In some embodiments,binding of the aptamer and the target is detected using the non-limitingexamples of SPR, RlfS, BLI, LFD or ELONA as described herein.

In some embodiments, the aptamer can be attached to the surface of thebiosensor using a biotin group. In some embodiments, the biotin group isattached at the 5′ end or the 3′ end of the aptamer. In someembodiments, the surface of the biosensor has an avidin/streptavidinattached thereto and the immobilization of the aptamer to the surface ofthe biosensor is via biotin-avidin interaction. In some embodiments, thesurface of the biosensor is coated with avidin/streptavidin.

Kits

Embodiments also provide a kit for detecting and/or quantifying C.difficile, wherein the kit comprises one or more aptamers as describedherein. Typically, the kit also comprises a detectable molecule asdescribed herein.

Embodiments provide a kit that further comprises a light source asdescribed herein. In an embodiment, the kit may further comprise abandpass filter as described herein. In an embodiment, the kit maycomprise viewing goggles or glasses or the like as described herein. Insome embodiments, the kit comprises:

-   -   a) A solution comprising aptamers having a detection molecule        conjugated thereto e.g. a fluorophore capable of emitting at a        wavelength of between about 485-515 nm. In some embodiments, the        fluorophore is capable of emitting at a wavelength of between        about 490-505 nm. In an embodiment the fluorophore is capable of        emitting at a wavelength of about 505 nm;    -   b) A light source. In some embodiments, the light source        produces light having a wavelength of between about 485-515 nm.        In an embodiment, the light source produces light having a        wavelength of between about 490-505 nm;    -   c) A bandpass filter. In an embodiment, the bandpass filter is a        590 nm bandpass filter; and    -   d) Viewing goggles. In an embodiment, the viewing goggles are        orange viewing goggles.

In some embodiments, the kit further comprises instructions for use inaccordance with any of the methods described herein.

The kit may comprise further components for the reaction intended by thekit or the method to be carried out, for example components for anintended detection of enrichment, separation and/or isolationprocedures. Non-limiting examples include buffer solutions, substratesfor a colour reaction, dyes or enzymatic substrates. In the kit, theaptamer may be provided in a variety of forms, including but not limitedto being pre-immobilized onto a support (e.g. solid support),freeze-dried, or in a liquid medium.

A kit herein may be used for carrying out any method described herein.It will be appreciated that the parts of the kit may be packagedindividually in vials or in combination in containers or multi-containerunits. Typically, manufacture of the kit follows standard procedureswhich are known to the person skilled in the art.

Uses

In some embodiments, method of detecting C. difficile, e.g. C. difficilespores, using the aptamers described herein, is provided. The method maycomprise interacting the sample with an aptamer described herein anddetecting the presence, absence, and/or amount of Clostridium difficile.The method may be for detecting the presence, absence, and/or amount ofClostridium difficile spores in a sample using a detection methodincluding, but not limited to, photonic detection, electronic detection,acoustic detection, electrochemical detection, electro-optic detection,enzymatic detection, chemical detection, biochemical detection, orphysical detection.

In some embodiments, the method is for detecting the presence, absence,or amount of C. difficile, e.g. C. difficile spores, on a surface. Insome embodiments, the aptamers and method provided may have utility indetecting C. difficile on surfaces in hospital and healthcarefacilities. Non-limiting examples of surfaces may include bed linen,medical equipment, clothing, floors, walls, and the like. In anon-limiting example, the aptamers of the present invention may be usedto detect the presence, absence, and/or amount of C. difficile, e.g. C.difficile spores, on a patient's body.

In some embodiments, the aptamers may be for use in detecting C.difficile, e.g. C. difficile spores, in a sample previously obtainedfrom a surface as described herein.

In some embodiments, the aptamers of the invention may be used to detectwhole C. difficile spores. In some embodiments, the aptamers may be usedto detect C. difficile proteins as described herein.

In some embodiments, the aptamers may be used to detect C. difficilespores or proteins in real-time. Following detection and/orquantification of C. difficile, action may be taken to kill and/orremove the spores. Non-limiting examples of such action may includewashing or destruction of bed linen, and/or cleaning of surfacesincluding but not limited to medical equipment, beds, walls, floors, andthe like. Measures such as isolation of patients and enforcement ofstringent hygiene protocols may also be taken.

In some embodiments, the aptamers of the invention are for use in amethod of detecting the presence or absence of C. difficile spores usinga light source. In certain embodiments, there is provided a method ofdetecting the presence or absence of C. difficile spores comprising:

-   -   a) Providing an aptamer conjugate comprising an aptamer        described herein, wherein the aptamer is conjugated to a        detectable moiety. In an embodiment, the detectable moiety is a        fluorescent moiety;    -   b) Contacting the aptamer conjugate with a location of interest,        wherein the location of interest may comprise C. difficile        spores;    -   c) Incubating the aptamer conjugate at the location of interest        for a predetermined period of time to allow the aptamer        conjugate to bind to a C. difficile spore if present;    -   d) Optionally washing the location of interest to remove any        unbound aptamer conjugates; and    -   e) Visualizing the aptamer conjugate bound to a C. difficile        spore.

In some embodiments, the location comprises a surface. In someembodiments, the location comprises a human, e.g. a patient's body, or asample obtained from a subject suspected of having or diagnosed with aClostridium difficile infection. In some embodiments, the locationcomprises an object located in a hospital environment

In some embodiments, visualizing the aptamer conjugate comprisesilluminating the location with a light source. In some embodiments, thelight source produces light at a predetermined wavelength, wherein thepredetermined wavelength corresponds to a wavelength of light emitted bythe detectable moiety of the aptamer conjugate.

In some embodiments, the step of visualizing the location may beperformed in ambient light or in dark conditions.

In some embodiments, the method further comprises filtering the lightproduced by the light source.

In some embodiments, the method further comprises imaging (e.g.photographing) the location and detecting the presence or absence of C.difficile spores.

In some embodiments, the method of detecting C. difficile may compriseapplying one or more of the aptamers of the invention to a locationsuspected of comprising C. difficile spores. Following a predeterminedperiod of time sufficient to permit the aptamer binding to C. difficilespores, the location may be washed one or more times to remove anyunbound aptamer. The method may then comprise a set of conditions forilluminating the location using a light source. In an embodiment, thelight source may be in the form of a forensic light source. In anembodiment, the light source may be in the form of a Polilight® Flare.

In some embodiments, the light source may be capable of switchingbetween different wavelengths, each wavelength being suited to aspecific interchangeable filter. The forensic light source may be in theform of a LED, laser, Polilight® or the like. In some embodiments, thelight source is a handheld light source. In an embodiment, the handheldlight source may be a Polilight Flare+2, which is a battery operated,handheld LED light source, available from e.g. Rofin Forensic.

Aptly, each Polilight Flare “torch” may produce light within a specifiedwavelength range. For example, in some embodiments, the light source mayproduce light at a wavelength of between about 360 nm-385 nm (UV light).In some embodiments, the light source may produce light at a wavelengthof between about 405 nm-420 nm. In some embodiments, the light sourcemay produce light at a wavelength of between about 435 nm-4.65 nm. Insome embodiments, the light source may produce light at a wavelength ofbetween about 485 nm-515 nm. In some embodiments, the light source mayproduce light at a wavelength of between about 490 nm-505 nm. In someembodiments, the light source may produce light at a wavelength ofbetween about 510 nm-545 nm. In some embodiments, the light source mayproduce light at a wavelength of between about 530 nm-560 nm. In someembodiments, the light source may produce light at a wavelength ofbetween about 585 nm-605 nm. In some embodiments, the light source mayproduce light at a wavelength of between about 615 nm-635 nm. In someembodiments, the light source may produce light at a wavelength ofbetween about 400 nm-700 nm. In some embodiments, the light source mayproduce light at a wavelength of between about 835 nm-865 nm. In someembodiments, the light source may produce light at a wavelength ofbetween about 935 nm-965 nm.

In some embodiments, the light source used may be compatible with adetectable molecule conjugated to the aptamer. In some embodiments, theaptamer is conjugated to a detection molecule. In some embodiments, thedetection molecule may be a fluorophore which emits in a spectral rangewhich corresponds to the output of the light source. In someembodiments, the aptamer may be conjugated to a fluorophore which emitsat a wavelength of about 505 nm. In some embodiments, the light sourceproduces light having a wavelength of about 505 nm.

In some embodiments, the method may comprise the use of a bandpassfilter in combination with the light source. The bandpass filter may beconfigured to transmit light of a certain wavelength band and rejectstray light outside the predetermined wavelength band. In someembodiments, the light source is configured to produce narrow bands oflight having centre wavelengths of 365 nm, 415 nm, 450 nm, 505 nm, 530nm, 545 nm, 620 nm, and 850 nm. In some embodiments, the light source isconfigured to produce narrow bands of light having a center wavelengthof 505 nm, in addition to white light wavelengths. In some embodiments,the bandpass filter is a 590 nm bandpass filter.

In some embodiments, the method may further comprise visualizing thelocation with viewing goggles, glasses, or the like. In someembodiments, the viewing goggles are of a colour which corresponds tothe colour of light produced by the light source and emitted by thedetection molecule conjugated to the aptamer. In some embodiments, thegoggles are orange and thus are suitable for use in combination with alight source which produces light having a wavelength of between about485 nm-515 nm, e.g. 505 nm, and an aptamer which comprises a detectionmolecule that emits at a wavelength of approximately 505 nm.

In an aspect, the invention relates to the development of aptamers whichbind to Clostridium difficile and methods of using the same. In anaspect, the invention relates to aptamers which specifically bind to aC. difficile spore. The aptamers may specifically bind to a C. difficileprotein; e.g. a surface protein. The molecule that an aptamer binds tomay be referred to as a target molecule. Further details of the targetmolecules are provided herein.

Unexpectedly, the present inventors have identified aptamers which arecap able of identifying C. difficile spores.

In embodiments, the invention provides an aptamer capable ofspecifically binding to a Clostridium difficile protein.

In embodiments, the Clostridium difficile protein is a surface proteinof Clostridium difficile spore. In embodiments, the Clostridiumdifficile protein is a spore coat surface protein or an exosporium layerprotein.

In embodiments the Clostridium difficile protein selected from CdeC,CdeM, CotA, CotE and CotE Chitinase.

In embodiments the Clostridium difficile protein is a CdeC proteinhaving an amino acid sequence as set forth in SEQ ID NO 18.

In embodiments the Clostridium difficile protein is a CdeM proteinhaving an amino acid sequence as set forth in SEQ ID NO: 19.

In embodiments the Clostridium difficile protein is a CotA, proteinhaving an amino acid sequence as set forth in SEQ ID NO: 15.

In embodiments the Clostridium difficile protein is a CotE, proteinhaving an amino acid sequence as set forth in SEQ ID NO: 16.

In embodiments the Clostridium difficile protein is a CotE Chitinaseprotein having an amino acid sequence as set forth in SEQ ID NO: 17.

In embodiments the aptamer comprises or consists of:

-   -   a) a nucleic acid sequence selected from any one of the nucleic        acid sequences as set forth in any of SEQ ID NOs: 1 to 14;    -   b) a nucleic acid sequence having at least 85%, for example 90%,        95%, 96%, 97%, 98%, or 99% identity with any one of the nucleic        acid sequence the nucleic acid sequences as set forth in any of        SEQ ID NOs: 1 to 14;    -   c) a nucleic acid sequence having at least about 30 consecutive        nucleotides of any one the nucleic acid sequences as set forth        in any of SEQ ID NOs: 1 to 14;    -   d) a nucleic acid sequence having at least about 30 consecutive        nucleotides of a sequence having at least 85% identity with any        one of SEQ ID NOs: 1 to 14;    -   e) a nucleic acid sequence having a fragment extending from        position 28 to position 64 of SEQ ID NO: 5, also known as SEQ ID        NO: 23; or    -   f) a nucleic acid sequence having a fragment extending from        position 28 to position 64 of SEQ ID NO: 5, also known as SEQ ID        NO: 23 having at least 85% identity with SEQ ID NO: 23.

In embodiments the aptamer is a single stranded DNA aptamer.

In embodiments, there is provided an aptamer that competes for bindingto a Clostridium difficile protein with the aptamer as described herein.

In embodiments the aptamer comprises a detectable label.

In embodiments the detectable label is and/or comprises a moietyselected from a fluorophore, a nanoparticle, a quantum dot, an enzyme, aradioactive isotope, a pre-defined sequence portion, a biotin, adesthiobiotin, a thiol group, an amine group, an azide, an aminoallylgroup, a digoxigenin, an antibody, a catalyst, a colloidal metallicparticle, a colloidal non-metallic particle, an organic polymer, a latexparticle, a nanofiber, a nanotube, a dendrimer, a protein, and aliposome. In some embodiments, the detectable label is a fluorophore, aquantum dot, a colloidal metallic particle, or a colloidal non-metallicparticle. In some embodiments, the detectable label is attached to anaptamer described herein via a moiety selected from a thiol group, anamine group, an azide and an aminoallyl group and combinations thereof.

In an aspect of the present invention, there is provided a complexcomprising an aptamer of any preceding claim and a detectable molecule.

In an aspect of the present invention, there is provided a compositioncomprising at least one aptamer, wherein at least one of the aptamers isas described herein wherein the composition optionally comprises atleast one of water, salts, one or more buffer herein, a detergent, andBSA.

In an aspect of the present invention, there is provided a compositioncomprising at least one aptamer having a nucleic acid sequence as setforth in SEQ ID NO: 1, 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 23wherein the composition optionally comprises at least one of water,salts, one or more buffer herein, a detergent, and BSA.

In an aspect of the present invention, there is provided a biosensor ortest strip comprising an aptamer as described herein.

In an aspect of the present invention, there is provided an apparatusfor detecting the presence, absence or level of Clostridium difficile ina sample, the apparatus comprising:

-   -   i. a support; and    -   ii. an aptamer as described herein.

In embodiments, the apparatus is for detecting the presence, absence orlevel of Clostridium difficile spores in a sample.

In embodiments, the sample is selected from:

-   -   a) a sample previously obtained from a subject suspected of        having or diagnosed with a Clostridium difficile infection; and    -   b) an object located in a hospital environment, for example        bedding, furniture, building structures.

In embodiments, the support is a bead, a microtiter or other assayplate, a strip, a membrane, a film, a gel, a chip, a microparticle, ananoparticle, a nanofiber, a nanotube, a micelle, a micropore, ananopore or a biosensor surface.

In embodiments, the apparatus is suitable for surface plasmon resonance(SPR), biolayer interferometry (BLI), lateral flow assay and/orenzyme-linked oligonucleotide assay (ELONA).

In an aspect of the present invention, there is provided a use of anaptamer a complex, a biosensor or test strip, a composition or apparatusas described herein for detecting, enriching, separating and/orisolating Clostridium difficile. In certain embodiments, the use is forspecifically detecting, enriching, separating and/or isolatingClostridium difficile spores.

In an aspect of the present invention, there is provided a method ofdetecting the presence, absence or amount of Clostridium difficile in asample, the method comprising:

-   -   i. interacting the sample with an aptamer, a complex, or a        composition as described herein; and    -   ii. detecting the presence, absence or amount of Clostridium        difficile.

In some embodiments, the method is for detecting the presence, absenceor amount of Clostridium difficile spores in a sample.

In some embodiments, the presence, absence or amount of Clostridiumdifficile is detected by photonic detection, electronic detection,acoustic detection, electrochemical detection, electro-optic detection,enzymatic detection, chemical detection, biochemical detection orphysical detection.

In an aspect of the present invention, there is provided a kit fordetecting and/or quantifying Clostridium difficile the kit comprising anaptamer as described herein.

EXAMPLES

In the following, the invention will be explained in more detail bymeans of non-limiting examples of specific embodiments. In the exampleexperiments, standard reagents and buffers free from contamination areused.

Example 1—Aptamer Selection

Target Information

Aptamers were selected against several protein targets of C. difficile.The targets were as follows:

1. CdeC, a protein which has a molecular weight (MW) of 46,000 Da.Stored in a buffer with the following composition:

20 mM HEPES-Na, pH 7.9, 5% glycerol, 200 mM NaCl, 0.2 mM CaCl₂, 0.1%Triton X114

Concentration: 0.75 mg ml⁻¹

2. CdeM a protein having a MW of 25,000 Da. Storage Buffer: 20 mMHEPES-Na, pH 7.9, 5% glycerol, 200 mM NaCl, 0.2 mM CaCl₂, 0.1% TritonX114

Concentration: 0.50 mg ml⁻¹

3. CdeM a protein with a MW of 25,000 Da. Storage Buffer: 20 mMHEPES-Na, pH 7.9, 5% glycerol, 200 mM NaCl, 0.2 mM CaCl₂, 0.1% TritonX114

Concentration: 0.50 mg ml⁻¹

4. CotA-His6 a protein with a MW of 34,900 Da Ext. Co: 27695 in water

Storage Buffer: 20 mM HEPES, 5% glycerol, 200 mM NaCl, 1 mM DTTConcentration: 4.17 mg ml⁻¹

5. rCotE, N281-F712, Molecular Weight: 48,000 Da,

Storage Buffer: 20 mM HEPES-Na, pH 7.9, 5% glycerol, 200 mM NaCl, 0.2 mMCaCl₂, 0.1% Triton X114

Concentration: ˜0.8 mg ml⁻¹

6. SPG-HU58. Non-pathogenic spores. Storage buffer: Sterile dH₂O

Concentration: 1×10⁷ CFU pure spores in 0.5 mL sterile water

Preparation for Aptamer Selection

The protein targets were each analyzed using a Nanodrop to generate aseries of UV spectra, to confirm concentration and aggregation state ofthe targets (data not shown). Analysis of the UV spectra for thesupplied CdeC, CdeM and CotE show clear signs of aggregation ormultimerization. CotA and CotEC Chitinase show slight signs ofaggregation. It is considered that some proteins may multimerize.

In addition, the targets were subjected to a ‘Buffer Screen’ with apanel of selection buffers. Binding of the aptamer library to beadsimmobilized with each target or blank beads were compared (data notshown). The buffer for each target which promoted greater interactionbetween the aptamer library and the target was identified and selectedfor future use in the selection process.

Non-limiting, exemplary buffers may be broadly similar for all of thetargets. In some embodiments, the buffer may be a Tris buffer. In someembodiments, the pH may be approximately 7.4 to 7.6. In someembodiments, the ionic strength may be approximately 100 mM.Non-limiting examples of salts included in the buffer are MgCl₂ andCaCl₂. In some embodiments, the buffer may comprise detergents,including but not limited to Tween. In some embodiments, the buffer maycomprise bovine serum albumin (BSA) or other stabilizers known in theart.

The buffers are as follows:

-   -   CdeC—50 mM Tris pH 7.6, 2.5 mM MgCl₂, 2.5 mM CaCl₂, 85 mM KoAc,        0.01% Tween 20, 0.01% BSA.    -   CdeM—50 mM Tris pH 7.6, 2.5 mM MgCl₂, 2.5 mM CaCl₂, 85 mM NaCl,        0.01% Tween 20, 0.01% BSA.    -   CotA—50 mM Tris pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 77.5 mM NaCl,        4.5 mM KCl, 0.01% Tween 20, 0.01% BSA.    -   CotE—50 mM Tris pH 7.6, 2.5 mM MgCl₂, 2.5 mM CaCl₂, 28 mM K₂SO₄,        0.01% Tween 20, 0.01% BSA    -   rCotE Chitinase—50 mM Tris pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 77.5        mM NaCl, 4.5 mM KCl, 0.01% Tween 20, 0.01% BSA.        Polyclonal Aptamer Selection

The selection protocol was broadly as followed:

His-tagged target protein were each loaded on to Ni-NTA coated magneticbeads and incubated for 1 hour in PBS. Loaded beads were washed andquantified, and used in aptamer selection.

The aptamer library was incubated with respective targets for one hourwith constant mixing at room temperature in a selection buffer asidentified in the buffer screen and shown above.

Target protein and bound aptamers were eluted using imidazole. Recoveredmaterial was subsequently purified to remove imidazole and amplified tocreate the enriched library for the subsequent selection round.

The process was repeated using increasing stringency from one selectionround to the next.

The buffer conditions identified in the upfront screens were used forthe first two rounds of in vitro selection. Subsequent rounds wereconducted using a variety of different selection ‘pressures’. Thepopulation from the best performing condition in each selection roundwas taken forward to the subsequent selection round. The amount ofaptamer recovered during selection is quantified and is shown in FIGS.6-10.

FIGS. 6-10 show the aptamer library recovery from the target-loadedbeads (blue, on left side of each data set) gradually increases withsequential rounds of selection. Any fall in recovery generally coincideswith the introduction of an increase in stringency during that round ofselection. The best target: negative ratio (recovery from target-loadedbeads vs. recovery from blank beads) was obtained in round 7 (R7) fortargets CotA, CdeC, CdeM and CotEC Chitinase, and in R10 for targetCotE, respectively. Each of these aptamer populations was then takenforward to a biophysical assay to confirm enrichment of target bindingspecies.

Biophysical Characterization

Biolayer Interferometry (BLI) was used to assess the binding of eachaptamer population to their respective targets. The target proteins wereimmobilized on separate Biolayer Interferometry sensor probes. Theloaded probes were then incubated with the naïve aptamer library or therespective aptamer populations to monitor and compare the interactions.

BLI was performed at room temperature using the same buffer as thoseused during the selection. BLI probes were loaded with target protein in1×PBS for 180 seconds. Subsequently the naïve aptamer library orrespective aptamer populations were incubated for 300 seconds. Theaptamers were then dissociated for 300 seconds in selection buffer.

The results are presented below in FIGS. 11-15.

FIGS. 11-15 show that the refined aptamer populations that haveundergone the aptamer selection process described herein generally haveimproved binding to their respective targets compared to the unselectednaïve library (some better than others). The immobilized targets showlittle to no interaction with unrefined naïve aptamer population.Binding is seen between the immobilized targets and the respectiverefined aptamer populations. Rapid association of the respective aptamerpool is seen for immobilized CotA, CotE and CotEC Chitinase (signals at˜480-780 sec). Both aptamer pools for CdeC and CdeM showed slowerassociation to their respective targets. The bound aptamer populationsdo not appear to show significant dissociation from their targets(signals at 780-1080 sec).

Spore Selection

The refined aptamer populations described above were taken into‘spore-based selection’ using Clostridium difficile spores as ‘positivetarget.’ Bacillus subtilis spores were used as a ‘negative target’(counter selection) to reduce non-specific binding to spore surfaces.Four subsequent rounds of spore-based selection (rounds S1-S4) wereperformed. The amount of aptamer recovered during these selection roundsis quantified and shown in FIGS. 16-20.

After 4 consecutive rounds of spore-based selection (S1-S4); the fiveaptamer populations, selected against CotA, CdeC, CdeM, and CotE, allshowed enhanced binding to the Clostridium difficile spores (‘positive’)compared to Bacillus subtilis spores (‘negative’). This indicatedfurther refinement of each of the aptamer populations in the context ofthe spore ‘coat’.

Selectivity Profiling

The refined aptamer populations isolated against recombinant CotA, CdeC,CdeM, CotE and CotEC Chitinase, and subsequently further refined byspore-based selection; were fluorescently labelled and incubated witheither Clostridium difficile spores or Bacillus subtilis spores. Unboundmaterial was removed by washing, before imaging the spores byepifluorescence microscopy. The results are shown in FIGS. 21-25.

FIGS. 21-24 demonstrate that four of the isolated aptamer populationsappear to bind preferentially to the Clostridium difficile sporescompared to the Bacillus subtilis spores. These aptamer populationsincluded CotA, CdeC, CdeM and CotE, respectively.

Conclusion

The reported data shows the following:

-   -   Biolayer Interferometry shows that the refined aptamer        populations selected against CotA, CdeC, CdeM, CotE and CotEC        Chitinase proteins, interact with their respective immobilized        target. Interactions were a result of selection process (not        simply through non-specific binding) as the ‘Naïve’ population        did not show such interaction.    -   Epifluorescence microscopy showed that four of the aptamer        populations have preferential binding to Clostridium difficile        spores compared to Bacillus subtilis spores. These aptamer        populations were isolated against CotA, CdeC, CdeM and CotE and        subsequently refined by spore-based selection using C. difficile        spores (positive) and B. subtilis spores (negative).    -   Aptamer populations isolated for CotEC Chitinase showed binding        to both Clostridium difficile and Bacillus subtilis spores after        3 rounds of spore-based selection. No binding was seen for C.        difficile after the 4th spore-based selection round.

Example 2—Monoclonal Aptamer Isolation

The refined pools (characterized in FIGS. 21-25) were taken forwards formonoclonal isolation. All aptamers were purified (after elution) andresuspended and stored in water. Before use in selections or bindingassays, aptamers were diluted in a final concentration 1× buffer.Individual aptamer clones were isolated and screened by BLI usingaptamer concentrations of 0.5 μM, 1 μM, or 2 μM. Again, the target wasimmobilized onto a Biolayer Interferometry sensor probe and thenincubated with each aptamer clone. The results are presented below(FIGS. 26-30). Results for the clones that did not meet specificationsare excluded for clarity.

FIGS. 26-30 show that the selected monoclonal aptamers have improvedbinding to their respective targets compared to the unselected naïvelibrary. The immobilized targets showed little to no interaction withunrefined naïve aptamer population. Binding was seen between theimmobilized targets and the respective selected monoclonal aptamers.

Rapid association of the monoclonal aptamers was seen for immobilizedCdeM, CdeC, CotE and CotEC Chitinase (signals at ˜60-240 sec). The boundmonoclonal aptamers did not appear to show significant dissociation fromCotEC Chitinase, CotA and CdeC. There was a higher rate of dissociationof the monoclonal aptamer from CdeM and CotE for the specific monoclonalaptamer pools used in this example (signals at ˜240-420 sec); however,rapid association for their respective immobilized target proteinoccurred as described herein.

Both selected monoclonal aptamers for CotA showed slower association totheir respective target but very little dissociation of the boundaptamers.

The naïve library control for the CotE aptamers showed a slightassociation. This was considered to be an anomalous result that has noeffect on the integrity of the data.

Conclusion

Biolayer Interferometry shows that the selected monoclonal aptamersselected against CotA, CdeC, CdeM, CotE and CotEC Chitinase proteins,interact with their respective immobilized target. Interactions are aresult of selection process (not simply through non-specific binding) asthe ‘naïve’ population shows no such interaction.

Example 3—Detection

The ability of the Biovector CotE H2 aptamer to visualise Clostridiumdifficile SH11 bacterial spores on stainless-steel and gown surfaces, inambient light and dark conditions was assessed.

Materials and Methods

Clostridium difficile purified spore suspensions used in this study arelisted in Table 6. C. difficile suspensions were provided by SporeGen®and stored at 4° C. upon arrival.

TABLE 6 Test organisms In test concentration Ribotype Format Description(CFU mL-1) C. difficile RT078 Wild type Purified 1 × 107 ± (SH11) spore5 × 106 suspension

Test agents used throughout the study are described in Table 7. The CotEH2 aptamer and TbKst buffer were provided by Aptamer Group.

TABLE 7 Test agents In-test aptamer Test agent concentration name FormatDescription (μM) Negative Solid Stainless-steel or N/A control 1 surfacegown surface only Negative Liquid C. difficile SH11 N/A control 2 sporesonly Negative Liquid Horse blood only N/A control 3 Positive Liquid CotEH2 aptamer 10 control 4 in TbKst buffer CotE H2 + Liquid CotE H2 aptamer10 SH11 in TbKst buffer incubated with C. difficile SH11 sporesEquipment:UKAS calibrated pipettes—Sartorius,UK Eppendorf 5452Minispin Centrifuge—Eppendorf, DEPolilight® Flare+2 forensic lights (wavelength 505 nm)—Rofin,UK Stainless-steel tableHospital gown surfaceCanon EOS 2000D camera—590 nm wavelength filter, Midwest Optical Systems, Inc.Media:Nuclease free water—provided by Aptamer GroupTbKst buffer—provided by Aptamer Group. The buffer solution contains 50mM Tris pH 7.6, 2.5 mM MgCl₂, 2.5 mM CaCl₂, 28 mM K₂SO₄, 0.01% Tween,0.01% BSA.Clinell sporicidal wipes—GAMA healthcare,UK 70% isopropyl alcohol (IPA)—Fisher Scientific, UK—1% Virkon solution—Scientific Laboratory Supplies, UKMethod

Assessment of the Ability of the Biovector CotE H2 Aptamer to DetectClostridium difficile SH11 Bacterial Spores on Stainless-Steel and GownSurfaces, in Ambient Light and Dark Conditions.

CotE H2 Aptamer and Clostridium difficile SH11 Binding Procedure:

Prior to testing, the CotE H2 aptamer was folded in nuclease free waterby heating to 95° C. for 5 minutes. The CotE H2 aptamer was immediatelycooled to 2° C. on ice. An inoculum of C. difficile SH11 bacterialspores was prepared to 1×107 CFU mL-1 from stock solution in nucleasefree water. Once folded, 20 μL of the C. difficile SH11 spore suspensionwas added to 20 μL of 20 μM of folded CotE 1-12 aptamer to obtain afinal concentration of 10 μM. The aptamers comprise a FAM fluorophoreincorporated at the 5′ end via a linker.

The aptamer-spore suspension was mixed and vortexed for 5 seconds toobtain a homogenous suspension and incubated for 1 hour at roomtemperature. Following incubation, the aptamer-spore suspension waswashed by centrifugation to remove unbound CotE H2 aptamer. One hundredmicroliters of TbKst buffer was added to the aptamer-spore suspensionand centrifuged at 12,100×g (13,000 rpm) for 10 minutes. The supernatantliquid was discarded. The aptamer-spore pellet was resuspended in 100 μLof TbKst buffer and vortexed for 10 minutes to obtain a homogenoussuspension. For negative control 4 (CotE 1-12 aptamer in TbKst bufferwithout spores), 10 μL of TbKst buffer was added to 10 μL of 20 μM offolded CotE H2 aptamer to obtain a final concentration of 10 μM.

Detection of Bacterial Spores from Clostridium difficile SH11 onStainless-Steel and Gown Surfaces in Ambient Light and Dark Conditions

A stainless-steel surface was cleaned sequentially with sporicidalwipes, 1% Virkon™ solution and 70% isopropyl alcohol (IPA). Followingcleaning, the surface was rinsed with water. The surface was dividedinto five 10×10 cm samples labelled S1-S5. Sample S1 was untreated toact as a clean surface control for the stainless-steel surface (negativecontrol 1). Five×5 μL aliquots of the negative control 2 (C. difficileSH11 spores only), negative control 3 (horse blood only), positivecontrol 4 (CotE H2 aptamer at 10 μM in TbKst buffer) and theaptamer-spore suspension were pipetted onto samples S2, S3, S4 and S5respectively. The surface was allowed to dry at room temperature for 1hour. Following drying, the fluorescence of the aptamer-spore suspensionwas assessed in ambient light and dark conditions, with and without thePolilight® Flare+2 forensic light (505 nm). Images of the fluorescencewere taken with a Canon EOS 2000D with and without a 590 nm bandpassfilter. Autofluorescence of the negative controls was also assessed. Thetest was repeated on hospital gown surfaces.

Results

Assessment of the Ability of the CotE H2 Aptamer to Detect Clostridiumdifficile SH11 Bacterial Spores on Stainless-Steel and Gown Surfaces, inAmbient Light and Dark Conditions:

Stainless-Steel Surface:

Ambient Light, without Polilight® Flare+2 Forensic and without 590 nmBandpass Filter

No visible fluorescence was observed for all the test samples withoutthe exposure to Polilight® Flare+2 forensic light (505 nm) and the 590nm bandpass filter in ambient light conditions (FIG. 31A-FIG. 31E).

Ambient Light, with Polilight® Flare+2 Forensic and without 590 nmBandpass Filter

Visible reflection of the Polilight® Flare+2 forensic light caused bythe stainless-steel surface was observed for test sample when exposed toPolilight® Flare+2 forensic light (505 nm) without the 590 nm bandpassfilter in ambient light conditions (FIG. 32A). Visible reflection of thetest sample was also observed within the samples containing C. difficileSH11 spores (FIG. 32B), horse blood (FIG. 32C), CotE H2 aptamer at 10 μM(FIG. 32D), and the combination of CotE H2 aptamer 10 μM and C.difficile SH11 spores (FIG. 32E).

Ambient Light, with Polilight® Flare+2 Forensic and with 590 nm BandpassFilter

No visible reflection of the Polilight® Flare+2 forensic light caused bythe stainless-steel surface was observed when exposed to Polilight®Flare+2 forensic light (505 nm) when using the 590 nm bandpass filter inambient light conditions (FIG. 33A-FIG. 33E). No visibleautofluorescence was observed on the samples containing thestainless-steel surface (FIG. 33A), C. difficile SH11 spores (FIG. 33B),or horse blood (FIG. 33C). Fluorescence was observed within the samplecontaining CotE H2 aptamer at 10 μM as illustrated by the bright lightin FIG. 33D (solid arrows). No visible fluorescence was observed withinthe sample containing the combination of CotE H2 aptamer 10 μM and C.difficile SH11 spores (FIG. 33E).

Dark Conditions, with Polilight® Flare+2 Forensic and with 590 nmBandpass Filter

Some visible reflection of the Polilight® Flare+2 forensic light causedby the stainless-steel surface was observed for all the test sampleswhen exposed to Polilight Flare+2 forensic light (505 nm) with the 590nm bandpass filter in dark conditions (FIG. 34A-FIG. 34E). Minimalautofluorescence was observed within the samples containing thestainless-steel surface (FIG. 34A), C. difficile SH11 spores (FIG. 34B),or horse blood (FIG. 34C). Some autofluorescence was also observed fromparticles present on the surface (indicated by dashed arrows, FIGS. 34,A, B, C and E). Fluorescence was observed on the stainless-steelsurface, within the sample containing CotE H2 aptamer at 10 μM asillustrated by the bright light in FIG. 34D (solid arrows). Fluorescencewas also observed with the sample containing the combination of CotE H2aptamer 10 μM and C. difficile SH11 spores (FIG. 34E; solid arrows).

Gown Surface

Ambient Light, without Polilight® Flare+2 Forensic and without 590 nmBandpass Filter

No fluorescence was observed for the gown surface test samples withoutthe exposure to Polilight® Flare+2 forensic light (505 nm) and the 590nm bandpass filter in ambient light conditions (FIG. 35A-FIG. 35E).

Ambient Light, with Polilight® Flare+2 Forensic and without 590 nmBandpass Filter

No visible fluorescence was observed for the test samples on the gownsurface when exposed to Polilight® Flare+2 forensic light (505 nm) whenobserved without the 590 nm bandpass filter in ambient light conditions(FIG. 36A-FIG. 36E). Visible reflection of the Polilight® Flare+2forensic light caused by the gown surface was observed for test sampleswhen exposed to Polilight® Flare+2 forensic light (505 nm) without the590 nm bandpass filter in ambient light conditions (FIG. 36A). Visiblereflection of test samples was also observed within the samplescontaining C. difficile SH11 spores (FIG. 36B), horse blood (FIG. 36C),CotE H2 aptamer at 10 μM (FIG. 36D), and the combination of CotE H2aptamer 10 μM and C. difficile SH11 spores (FIG. 36D).

Ambient Light, with Polilight® Flare+2 Forensic and with 590 nm BandpassFilter

Bright green/yellow fluorescence was observed within the samplecontaining CotE H2 aptamer at 10 μM (FIG. 37D; solid arrows) and,visible fluorescence was observed within the sample containing thecombination of CotE H2 aptamer 10 μM and C. difficile SH11 spores (FIG.37E; solid arrows). No visible reflection of the Polilight® Flare+2forensic light was observed on the gown surfaces when they were exposedto Polilight® Flare+2 forensic light (505 nm) and observed with the 590nm bandpass filter in ambient light conditions (FIG. 37A-FIG. 37E).

Dark Conditions, with Polilight® Flare+2 Forensic and with 590 nmBandpass Filter

Bright fluorescence was observed within the sample containing CotE H2aptamer at 10 μM (FIG. 38D; solid arrows). Fluorescence was observedwithin the sample containing the combination of CotE H2 aptamer 10 μMand C. difficile SH11 spores (FIG. 38E; solid arrows). Noautofluorescence was observed within the samples containing the gownsurface, C. difficile SH11 spores or horse blood (FIGS. 38A-38C).

Discussion

C. difficile is an anaerobic spore-forming microorganism and isconsidered a leading cause of infections worldwide, with elevated ratesof morbidity. A method of visual identification of C. difficile sporecontamination in the health care environment would allow improvedcleaning procedures.

The assessment of fluorescence for the stainless-steel showed thatfluorescence was detected only under dark conditions. The assessment offluorescence for the gown surface showed that fluorescence of the CotEH2 aptamer in combination with C. difficile SH11 spores was detectedunder both ambient light and dark conditions. The intensity of thefluorescence observed on the gown surface in response to the presence ofthe combination CotE H2 aptamer and C. difficile SH11 spores underambient light conditions was lower than intensity of the fluorescenceunder dark conditions. No visible reflection or autofluorescence wasdetected on the gown surface controls, but a high amount of reflectionwas observed on stainless-steel samples.

Example 4—Minimal Binding Fragments

A fragment of the chitinase_D11 aptamer (SEQ ID NO: 5) was found to bindto C. difficile spores. The fragment extended from position 28 toposition 64 of SEQ ID NO: 5. This sequence is also shown in SEQ ID NO:23. Binding was identified using an ELISA-type assay in which abiotinylated fragment of SEQ ID NO: 23 was used in a similar manner to aprimary antibody. A streptavidin-HRP conjugate was used as a secondarybinding partner.

Several fragments of the C. diff-F1 aptamer (SEQ ID NO: 1) were found tobind to C. difficile spores. The sequence for the minimal fragment, C.diff-F1-f10, is 5′-CCATACTCAATGCTCTTACGATCCTCATCAACC-3′, and is 33 baseslong (SEQ ID NO: 24). The fragment comprises 23 contiguous nucleotidesextending from position 12 to position 35 of SEQ ID NO: 1.

Several fragments of the C. diff-G1 aptamer (SEQ ID NO: 2) were found tobind to C. difficile spores. The sequence for the minimal fragment, C.diff-G1-f6, is 5′-CCAGTGTAGACTACTCAATGCTCTTACGATCCTCATCAACC-3′, and is41 bases long (SEQ ID NO: 25). The fragment comprises 21 contiguousnucleotides extending from position 1 to position 21 of Seq ID NO: 2.

Several fragments of the CotE-H2 aptamer (SEQ ID NO: 11) were found tobind to C. difficile spores. The sequence for the minimal fragment,CotE-H2-f4, is 5′-AGTGTAGACTACTCAATGCGGCTGGCCACAGGTCAACC-3′, and is 40bases long (SEQ ID NO: 26). The sequence comprises 24 contiguousnucleotides from the 5′ end and 13 contiguous nucleotides from the 3′end of SEQ ID NO: 11 (with an internal truncation of 42 nucleotides fromSEQ ID NO: 11) which were combined with an additional guanine bridge.

In a single point ELISA assay, the minimal fragments C. diff-F1-f10, C.diff-G1-f6, and CotE-H2-f4 each displayed preferential binding to sporesfrom C. diff. over B. sub. (approximately two-fold).

Example 5—Aptamer Binding to Proteins

The binding affinity, as reported by the equilibrium dissociationconstant (K_(D)), was determined for several aptamers and proteins(performed under contract by NeoVentures Biotechnology Inc. (London,Ontario, Canada)).

For CotE D2, CotE H2 and CotEC chitinase, aptamers were spotted on agold surface at a concentration of 5 μM in PBS at a volume ofapproximately 10 nL in triplicate. Negative aptamers of the same lengthas the positive aptamers were spotted in the same manner, also intriplicate. Protein was then injected over the chip at a volume of 200μL, with a flow rate of 50 μL/min in a Horiba (OpenPlex) surface plasmonresonance imaging (SPRi) instrument. Resonance due to binding wasobtained by subtracting total resonance on the negative sequences fromtotal resonance on the positive sequences. Disassociation values werecomputed with the following equation: dx/dt˜−kd*x, wherein dx/dt is thederivative of resonance values as a function of time and x is theresonance value for any given time point. Association values werecomputed with the following equation: dx/dt˜ka*c*Rmax−(ka*c+kd)*x, wherex is the resonance due to binding at specific time points, c is theconcentration of injectant, and Rmax is the maximum resonance observed.

For CotA C1, the protein was immobilized on a hydrogel chip withEDC-mediated conjugation between primary amines on the protein (sidechains on residues) and carboxylic acid groups on the surface of thechip. A protein of similar size was immobilized in the same manner as anegative control. The variation in concentration for this data is in theamount of aptamer injected. For CdeC D1, the protein was received asaggregated, insoluble balls of protein. The binding assay was performedby incubating fluorescently labeled aptamers (FAM) with the protein, andthen spinning the tubes down in a microcentrifuge to remove unbound. Thepellet was resuspended in selection buffer and spun down again. Boundaptamers were eluted by adding 6 M urea, and spinning again, retainingthe supernatant. The aptamers were cleaned up with PCR cleanup columnsand the fluorescence was read on a Tecan Sapphire II fluorometer.Excitation was at 497 nm, and emission was at 515 nm. The amount offluorescence measured in the eluant was divided by the total amount offluorescence of all fractions.

For aptamer CotE H2 (SEQ ID NO: 11), the K_(D) was determined to be1.43E-07 (at 250 nM protein), 9.16E-08 (at 125 nM protein), and 8.47E-08(at 62.5 nM protein), respectively. For aptamer CotE D2 (SEQ ID NO: 13),the K_(D) was determined to be greater than 250 nM. For aptamer CotA C1(SEQ ID NO: 10), the K_(D) was determined to be 6.874E-09. For aptamerCotEC Chitinase D11 (SEQ ID NO: 5), the K_(D) was determined to be2.54E-07 (at 500 nM protein), 2.35E-07 (at 750 nM protein), and 2.75E-07(at 1000 nM protein), respectively. For aptamer CdeC D1 (SEQ ID No 6),the K_(D) was not determined due to protein aggregation.

The references cited throughout this application, are incorporatedherein in their entireties for all purposes apparent herein and in thereferences themselves as if each reference was fully set forth. For thesake of presentation, specific ones of these references are cited atparticular locations herein. A citation of a reference at a particularlocation indicates a manner(s) in which the teachings of the referenceare incorporated. However, a citation of a reference at a particularlocation does not limit the manner in which all of the teachings of thecited reference are incorporated for all purposes.

It is understood, therefore, that this invention is not limited to theparticular embodiments disclosed but is intended to cover allmodifications which are within the spirit and scope of the invention asdefined by the appended claims; the above description; and/or shown inthe attached drawings.

What is claimed is:
 1. A method of visualizing Clostridium difficilespores on a surface, comprising: contacting a surface with an aptamerhaving a specific binding affinity for a surface protein of Clostridiumdifficile spore, wherein the aptamer comprises a nucleic acid sequencehaving at least 90% identity with the nucleic acid sequence as set forthin SEQ ID NO: 11, and wherein the surface protein is CotE; andvisualizing the presence or absence of C. difficile spores on thesurface.
 2. The method of claim 1, further comprising washing of thesurface after the contacting to remove unbound aptamer.
 3. The method ofclaim 1, wherein the aptamer is conjugated to a detectable moietythereby forming an aptamer conjugate.
 4. The method of claim 3, whereinthe detectable moiety is a fluorophore.
 5. The method of claim 4,wherein the fluorophore emits at a wavelength of between about 500 nmand 510 nm.
 6. The method of claim 4, further comprising illuminatingthe surface with a light source.
 7. The method of claim 6, wherein lightfrom the light source has a predetermined wavelength, and thepredetermined wavelength corresponds to a wavelength of light emitted bythe detectable moiety of the aptamer conjugate.
 8. The method of claim6, wherein the light source is configured to produce light at awavelength of between about 485 nm and 515 nm.
 9. The method of claim 6,further comprising filtering the light produced by the light source. 10.The method of claim 6, comprising passing the light produced from thelight source through a bandpass filter.
 11. The method of claim 10,further comprising photographing a location on the surface, anddetecting the presence or absence of the conjugated aptamer bound toClostridium difficile spores.